Methods and compositions for screening and treating developmental disorders

ABSTRACT

This document provides methods and materials related to genetic variations of developmental disorders. For example, this document provides methods for using such genetic variations to assess susceptibility of developing Autism Spectrum Disorder.

CROSS REFERENCE

This application is a divisional of U.S. Nonprovisional application Ser.No. 15/279,012, filed Sep. 28, 2016, which is a continuation applicationof U.S. Nonprovisional application Ser. No. 14/039,770, now U.S. Pat.No. 10,233,495 filed Sep. 27, 2013, issued Mar. 19, 2019, which claimsbenefit of U.S. Provisional Application No. 61/744,463, filed Sep. 27,2012, each of which is incorporated herein by reference in its entirety.

REFERENCE TO THE SEQUENCE LISTING

Three copies of the sequence listing (“Copy 1 of 3— Sequence Listing”;Duplicate “Copy 2 of 3— Sequence Listing” and Duplicate “Copy 3 of 3—Sequence Listing”) and a computer readable form (CRF copy) of thesequence listing, all in ASCII format created on Feb. 3, 2020 and named33655-708.403 Sequence listing.txt and is 594419.08 kilobytes in size.

BACKGROUND OF THE DISCLOSURE

Genetic risk can be conferred by subtle differences in individualgenomes within a population. Genes can differ between individuals due togenomic variability, the most frequent of which are due to singlenucleotide polymorphisms (SNPs). SNPs can be located, on average, every500-1000 base pairs in the human genome. Additional geneticpolymorphisms in a human genome can be caused by duplication, insertion,deletion, translocation and/or inversion, of short and/or long stretchesof DNA. Thus, in general, genetic variability among individuals occurson many scales, ranging from single nucleotide changes, to gross changesin chromosome structure and function. Recently, many copy numbervariations (CNVs) of DNA segments, including deletions, insertions,duplications, amplifications, and complex multi-site variants, rangingin length from kilobases to megabases in size, have been discovered(Redon, R. et al. Nature 444:444-54 (2006) and Estivill, X. & Armengol,L. PLoS Genetics 3(10): e190 (2007)). To date, known CNVs account forover 15% of the assembled human genome (Estivill, X. Armengol, L. PLoSGenetics 3(10): e190 (2007)). However, a majority of these variants areextremely rare and cover a small percentage of a human genome of anyparticular individual.

Today, it is estimated that one in every 88 children is diagnosed withAutism Spectrum Disorder (ASD) according to the CDC, making it morecommon than childhood cancer, juvenile diabetes and pediatric AIDScombined. An estimated 1.5 million individuals in the U.S. and tens ofmillions worldwide are affected by autism. Government statistics suggestthe prevalence rate of autism is increasing 10-17 percent annually.There is no established explanation for this increase, although improvedscreening and environmental influences are two reasons often considered.Studies suggest boys are five times more likely than girls to developautism and receive the screening three to four times more frequently.Current estimates are that in the United States alone, one out of 54boys is diagnosed with autism. ASD can be characterized by problems andsymptoms in the following areas: communication, both verbal andnon-verbal, such as pointing, eye contact, and smiling; social, such assharing emotions, understanding how others think and feel, and holding aconversation; and routines or repetitive behaviors (also calledstereotyped behaviors), such as repeating words or actions, obsessivelyfollowing routines or schedules, and playing in repetitive ways. Asgenetic variations conferring risk to developmental disorders, includingASD, are uncovered, genetic testing can play a role for clinicaltherapeutics.

Despite these advances towards an understanding of the etiology ofdevelopmental disorders, a large fraction of the genetic contribution tothese disorders remains undetermined. Identification of underlyinggenetic variants that can contribute to developmental disorderpathogenesis can aid in the screening and identification of individualsat risk of developing these disorders and can be useful for diseasemanagement. There is a need to identify new treatments for developmentaldisorders, specifically ASD, and the identification of novel geneticrisk factors and causes can assist in the development of potentialtherapeutics and agents. There is also a need for improved assays forpredicting and determining potential treatments and their effectiveness.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.In the event of a conflict between a term herein and a term incorporatedby reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings.

FIG. 1 represents an example of group 1 (Genic (distinctCNV-subregions); OR >6). There are 10 ASD cases and 0 NVE subjectsaffected by non-overlapping and overlapping CNV-subregions. The CNV aregains (log 2ratio >0.35) or losses (log 2ratio <−0.35) and affect thegene NRG1 on chromosome 8. The calculated odds ratio (OR) for thisCNV-subregion is 14.94.

FIG. 2 represents an example of group 2 (Exon+ve, ASD >4, Normals <2, noSanger filter applied). There are 34 ASD cases in total (including 31with an identical sized loss) and 1 NVE subject affected by overlappingCNV-subregions that impact an exon. The CNV are a gain (log2ratio >0.35) or losses (log 2ratio <−0.35) and affect the gene MIDN onchromosome 19. The calculated odds ratio (OR) for this CNV-subregion is52.68.

FIG. 3 represents an example of group 3 (Exon+ve, 5>ASD >1, Normals <2,Sanger −ve). There are 4 ASD cases in total and 1 NVE subject affectedby an overlapping CNV-subregion that impacts an exon. The CNV are losses(log 2ratio <−0.35) and affect the gene PTGER3 on chromosome 1 and noSanger CNVs overlap this CNV (Sanger −ve). The calculated odds ratio(OR) for this CNV-subregion is 5.92.

FIG. 4 represents an example of group 4 (Intron+ve, ASD >4, Normals <2,no Sanger filter applied). There are 8 ASD cases in total (3 casesimpact an identical CNV loss) and 0 NVE subjects affected by anoverlapping CNV-subregion that impacts an intron. The CNV are losses(log 2ratio <−0.35) or a gain (log 2ratio >0.35) and affect the geneCALN1 on chromosome 7. The calculated odds ratio (OR) for thisCNV-subregion is 11.92.

FIG. 5 represents an example of group 5 (MTRNR2L_family). There is 1 ASDcase and 0 NVE subjects that impacts an exon of an MTRNR2L gene familymember. The CNV gain (log 2ratio >0.35) is 1.7 Mb in size and its leftbreakpoint disrupts MTRNR2L4 and its right breakpoint disrupts ALG1 onchromosome 16. The calculated odds ratio (OR) for this CNV-subregion is1.47.

FIG. 6 represents an example of group 6 (High OR intergenic (OR >30)).There are 20 ASD cases in total (5 representative cases are depicted)and 0 NVE subjects affected by an overlapping CNV-subregion that impactsan intergenic region (adjacent to SDC1). The CNV are losses (log 2ratio<−0.35) on chromosome 2. The calculated odds ratio (OR) for thisCNV-subregion is 30.33.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a method of screening one or moresubjects for at least one genetic variation that disrupts or modulatesone or more genes in Table 3, comprising: assaying at least one nucleicacid sample obtained from each of the one or more subjects for the atleast one genetic variation in one or more genes in Table 3. In someembodiments, the at least one genetic variation is associated with adevelopmental disorder (DD). In some embodiments, the at least onegenetic variation is one encoded by one or more of SEQID NOs 1 to 883.In some embodiments, the at least one genetic variation comprises one ormore point mutations, single nucleotide polymorphisms (SNPs), singlenucleotide variants (SNVs), translocations, insertions, deletions,amplifications, inversions, microsatellites, interstitial deletions,copy number variations (CNVs), or any combination thereof. In someembodiments, the at least one genetic variation disrupts or modulatesone or more genes in Table 3. In some embodiments, the at least onegenetic variation disrupts or modulates two or more genes in Table 3. Insome embodiments, the at least one genetic variation disrupts ormodulates the expression or function of one or more RNA transcriptsencoded by SEQ ID NOs 884-1690, one or more polypeptides producedtherefrom, or a combination thereof. In some embodiments, the assayingcomprises detecting nucleic acid information from the at least onenucleic acid sample. In some embodiments, the nucleic acid informationis detected by one or more methods selected from the group comprisingPCR, sequencing, Northern blots, or any combination thereof. In someembodiments, the sequencing comprises one or more high-throughputsequencing methods. In some embodiments, the one or more high throughputsequencing methods comprise Massively Parallel Signature Sequencing(MPSS), polony sequencing, 454 pyrosequencing, Illumina sequencing,SOLiD sequencing, ion semiconductor sequencing, DNA nanoball sequencing,heliscope single molecule sequencing, single molecule real time (SMRT)sequencing, RNAP sequencing, Nanopore DNA sequencing, sequencing byhybridization, or microfluidic Sanger sequencing. In some embodiments,the at least one nucleic acid sample is collected from blood, saliva,urine, serum, tears, skin, tissue, or hair from the one or moresubjects. In some embodiments, the assaying the at least one nucleicacid sample of the one or more subjects comprises purifying nucleicacids from the at least one nucleic acid sample. In some embodiments,the assaying the at least one nucleic acid sample of the one or moresubjects comprises amplifying at least one nucleotide sequence in the atleast one nucleic acid sample. In some embodiments, the assaying the atleast one nucleic acid sample for at least one genetic variationcomprises a microarray analysis of the at least one nucleic acid sample.In some embodiments, the microarray analysis comprises a CGH arrayanalysis. In some embodiments, the CGH array detects the presence orabsence of the at least one genetic variations. In some embodiments, themethod further comprises determining whether the one or more subjectshas a DD, or an altered susceptibility to a DD. In some embodiments, theone or more subjects were previously diagnosed or are suspected ashaving the DD. In some embodiments, the diagnosis or grounds forsuspicion that the subject may have the DD is based on an evaluation bya medical doctor, a psychologist, a neurologist, a psychiatrist, orother professionals who screen subjects for the DD. In some embodiments,the determining comprises an evaluation of the one or more subject'smotor skills, autonomic function, neurophychiatry, mood, cognition,behavior, thoughts, speech, or a combination thereof. In someembodiments, the evaluation comprises observation, a questionnaire, achecklist, a test, or a combination thereof. In some embodiments, theevaluation comprises a developmental exam, the subject's past medicalhistory, or a combination thereof. In some embodiments, the screeningthe one or more subjects further comprises selecting one or moretherapies based on the presence or absence of the one or more geneticvariations. In some embodiments, the assaying at least one nucleic acidsample obtained from each of the one or more subjects comprisesanalyzing the whole genome or whole exome from the one or more subjects.In some embodiments, the nucleic acid information has already beenobtained for the whole genome or whole exome from the one or moreindividuals and the nucleic acid information is obtained from in silicoanalysis. In some embodiments, the DD is Autism Spectrum Disorder (ASD).In some embodiments, the one or more subjects have at least one symptomof a DD. In some embodiments, the at least one symptom comprisesdifficulty with verbal communication, including problems using andunderstanding language, difficulty with non-verbal communication, suchas gestures and facial expressions such as smiling, difficulty withsocial interaction, including relating to people and to his or hersurroundings, unusual ways of playing with toys and other objects,difficulty adjusting to changes in routine or familiar surroundings,repetitive body movements or patterns of behavior, such as handflapping, spinning, and head banging, changing response to sound, tempertantrums, difficulty sleeping, aggressive behavior, fearfulness,anxiety, or a combination thereof. In some embodiments, the one or moresubjects are human. In some embodiments, the one or more subjects areless than 30 years old, less than 20 years old, less than 10 years old,less than 5 years old, less than 2 years old, or less than 1 year old.

In one aspect, provided herein is a method of diagnosing one or morefirst subjects for a DD, comprising: assaying at least one nucleic acidsample of each of the one or more subjects for the presence or absenceof at least one genetic variation in one or more genes in Table 3. Insome embodiments, the at least one genetic variation is one encoded byat least one of SEQ ID NOs 1-883. In some embodiments, the one or morefirst subjects is diagnosed with the DD if the at least one geneticvariation is present. In some embodiments, the one or more firstsubjects is not diagnosed with DD if the at least one genetic variationis absent. In some embodiments, the assaying comprises detecting nucleicacid information from the at least one nucleic acid sample. In someembodiments, the nucleic acid information is detected by one or moremethods selected from the group comprising PCR, sequencing, Northernblots, hybridization, or any combination thereof. In some embodiments,the sequencing comprises one or more high-throughput sequencing methods.In some embodiments, the one or more high throughput sequencing methodscomprise Massively Parallel Signature Sequencing (MPSS), polonysequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing,ion semiconductor sequencing, DNA nanoball sequencing, heliscope singlemolecule sequencing, single molecule real time (SMRT) sequencing, RNAPsequencing, Nanopore DNA sequencing, sequencing by hybridization, ormicrofluidic Sanger sequencing. In some embodiments, the method furthercomprises determining whether the one or more first subjects has a DD oran altered susceptibility to a DD. In some embodiments, the one or morefirst subjects were previously diagnosed or are suspected as having theDD based on an evaluation by a medical doctor, a psychologist, aneurologist, a psychiatrist, a speech therapist, or other professionalswho screen subjects for a DD. In some embodiments, the determiningcomprises an evaluation of the one or more first subject's motor skills,autonomic function, neurophychiatry, mood, cognition, behavior,thoughts, speech, or a combination thereof. In some embodiments, theevaluation comprises observation, a questionnaire, a checklist, a test,or a combination thereof. In some embodiments, the evaluation comprisesa developmental exam, the subject's past medical history, or acombination thereof. In some embodiments, the determining comprisescomparing the nucleic acid information of the one or more first subjectsto nucleic acid information of one or more second subjects. In someembodiments, the one more second subjects comprise one or more subjectsnot suspected of having the DD. In some embodiments, the one or moresecond subjects comprise one or more subjects suspected of having theDD. In some embodiments, the one or more first subjects comprise one ormore subjects with the DD. In some embodiments, the one or more secondsubjects comprise one or more subjects without the DD. In someembodiments, the one or more first subjects comprise one or moresubjects who are symptomatic for the DD. In some embodiments, the one ormore second subjects comprise one or more subjects who are asymptomaticfor the DD. In some embodiments, the one or more first subjects compriseone or more subjects that have an increased susceptibility to the DD. Insome embodiments, the one or more second subjects comprise one or moresubjects that have a decreased susceptibility to the DD. In someembodiments, the one or more first subjects comprise one or moresubjects receiving a treatment, therapeutic regimen, or any combinationthereof for a DD. In some embodiments, determining whether the one ormore subjects have the DD or an altered susceptibility to the DDcomprises analyzing at least one behavioral analysis of the one or moresubjects and the nucleic acid sequence information of the one or moresubjects, or a combination thereof. In some embodiments, the at leastone nucleic acid sample is collected from blood, saliva, urine, serum,tears, skin, tissue, or hair from the one or more subjects. In someembodiments, assaying comprises purifying nucleic acids from the atleast one nucleic acid sample. In some embodiments, assaying comprisesamplifying at least one nucleotide sequence in the at least one nucleicacid sample. In some embodiments, assaying comprises a microarrayanalysis of the at least one nucleic acid sample. In some embodiments,wherein the microarray analysis comprises a CGH array analysis. In someembodiments, the CGH array detects the presence or absence of the atleast one genetic variations. In some embodiments, the at least onegenetic variation comprises one or more point mutations, singlenucleotide polymorphisms, (SNPs), single nucleotide variants (SNVs),translocations, insertions, deletions, amplifications, inversions,microsatellites, interstitial deletions, copy number variations (CNVs),or any combination thereof. In some embodiments, the at least onegenetic variation results in a loss of function for one or more genes inTable 3, a gain of function for one or more genes in Table 3, or acombination thereof. In some embodiments, the at least one geneticvariation disrupts or modulates the one or more genes in Table 3. Insome embodiments, the at least one genetic variation disrupts ormodulates the expression or function of one or more RNA transcriptsencoded by SEQ ID NOs 884-1690. In some embodiments, the method furthercomprises selecting one or more therapies based on the presence orabsence of the one or more genetic variations. In some embodiments, theassaying at least one nucleic acid sample obtained from each of the oneor more subjects comprises analyzing the whole genome or whole exomefrom the one or more subjects. In some embodiments, the nucleic acidinformation has already been obtained for the whole genome or wholeexome from the one or more individuals and the nucleic acid informationis obtained from in silico analysis. In some embodiments, the DD is ASD.In some embodiments, the one or more subjects has at least one symptomof a DD. In some embodiments, the at least one symptom comprisesdifficulty with verbal communication, including problems using andunderstanding language, difficulty with non-verbal communication, suchas gestures and facial expressions such as smiling, difficulty withsocial interaction, including relating to people and to his or hersurroundings, unusual ways of playing with toys and other objects,difficulty adjusting to changes in routine or familiar surroundings,repetitive body movements or patterns of behavior, such as handflapping, spinning, and head banging, changing response to sound, tempertantrums, difficulty sleeping, aggressive behavior, fearfulness,anxiety, or a combination thereof. In some embodiments, the one or moresubjects are human. In some embodiments, the one or more subjects isless than 30 years old, less than 20 years old, less than 10 years old,less than 5 years old, less than 2 years old, or less than 1 year old.

In one aspect, provided herein is a method of screening for atherapeutic agent for treatment of a DD, comprising identifying an agentthat disrupts or modulates one or more genes in Table 3, or one or moreexpression products thereof. In some embodiments, the one or moreexpression products comprise one or more RNA transcripts. In someembodiments, the one or more RNA transcripts comprise one or more RNAtranscripts of Table 4, or one ore more RNA transcripts encoded by anyof SEQ ID NOs 884-1690. In some embodiments, the one or more expressionproducts comprise one or more polypeptides. In some embodiments, the oneor more polypeptides are translated from one or more RNA transcripts ofTable 4, or one ore more RNA transcripts encoded by any of SEQ ID NOs884-1690. In some embodiments, disrupting or modulating the one or moregenes in Table 3 or one or more expression products thereof, comprisesan increase in expression of the one or more expression products. Insome embodiments, disrupting or modulating the one or more genes inTable 3 or one or more expression products thereof, comprises a decreasein expression of the one or more expression products.

In one aspect, provided herein is a method of treating a subject for aDD, comprising administering one or more agents to disrupt or modulateone or more genes in Table 3 or one or more expression products thereof,thereby treating the DD. In some embodiments, the one or more expressionproducts comprise one or more RNA transcripts. In some embodiments, theone or more RNA transcripts comprise one or more RNA transcripts ofTable 4, or one ore more RNA transcripts encoded by any of SEQ ID NOs884-1690. In some embodiments, the one or more expression productscomprise one or more polypeptides. In some embodiments, the one or morepolypeptides are translated from one or more RNA transcripts of Table 4,or one ore more RNA transcripts encoded by any of SEQ ID NOs 884-1690.In some embodiments, the one or more agents are selected from the groupcomprising: an antibody, a drug, a combination of drugs, a compound, acombination of compounds, radiation, a genetic sequence, a combinationof genetic sequences, heat, cryogenics, and a combination of two or moreof any combination thereof. In some embodiments, the DD is ASD. In someembodiments, the one or more subjects has at least one symptom of a DD.In some embodiments, the at least one symptom comprises difficulty withverbal communication, including problems using and understandinglanguage, difficulty with non-verbal communication, such as gestures andfacial expressions such as smiling, difficulty with social interaction,including relating to people and to his or her surroundings, unusualways of playing with toys and other objects, difficulty adjusting tochanges in routine or familiar surroundings, repetitive body movementsor patterns of behavior, such as hand flapping, spinning, and headbanging, changing response to sound, temper tantrums, difficultysleeping, aggressive behavior, fearfulness, anxiety, or a combinationthereof. In some embodiments, the one or more subjects is human. In someembodiments, the one or more subjects is less than 30 years old, lessthan 20 years old, less than 10 years old, less than 5 years old, lessthan 2 years old, or less than 1 year old.

In one aspect, provided herein is a kit for screening for a DD in one ormore subjects, the kit comprising reagents for assaying a nucleic acidsample from the one or more subjects for the presence of at least onegenetic variation encoded by SEQID NOs 1-883. In some embodiments, theat least one genetic variation disrupts or modulates one or more genesin Table 3, or one or more expression products thereof. In someembodiments, the one or more expression products comprise one or moreRNA transcripts. In some embodiments, the one or more RNA transcriptscomprise one or more RNA transcripts of Table 4, or one ore more RNAtranscripts encoded by any of SEQ ID NOs 884-1690. In some embodiments,the one or more expression products comprise one or more polypeptides.In some embodiments, the one or more polypeptides are translated fromone or more RNA transcripts of Table 4, or one ore more RNA transcriptsencoded by any of SEQ ID NOs 884-1690. In some embodiments, the reagentscomprise nucleic acid probes. In some embodiments, the reagents compriseoligonucleotides. In some embodiments, the reagents comprise primers. Insome embodiments, the DD is ASD. In some embodiments, the one or moresubjects has a symptom of a DD. In some embodiments, the one or moresubjects is human. In some embodiments, the one or more subjects is lessthan 30 years old, less than 20 years old, less than 10 years old, lessthan 5 years old, less than 2 years old, or less than 1 year old.

In one aspect, provided herein is an isolated polynucleotide sequence orfragment thereof, comprising at least 60% identity to any ofpolynucleotide sequence of SEQ ID NOs 1-1690. In one aspect, providedherein is an isolated polynucleotide comprising a CNV sequence encodedby any one of SEQ ID NOs 1-883. In some embodiments, the isolatedpolynucleotide sequence comprises at least 70% identity to any ofpolynucleotide sequence of SEQ ID NOs 1-1690. In some embodiments, theisolated polynucleotide sequence comprises at least 80% identity to anyof polynucleotide sequence of SEQ ID NOs 1-1690. In some embodiments,the isolated polynucleotide sequence comprises at least 90% identity toany of polynucleotide sequence of SEQ ID NOs 1-1690.

In one aspect, provided herein is an isolated polynucleotide sequencecomprising at least 60% identity to a compliment of any ofpolynucleotide sequence of SEQ ID NOs 1-1690. In some embodiments, theisolated polynucleotide sequence comprises at least 70% identity to acompliment of any of polynucleotide sequence of SEQ ID NOs 1-1690. Insome embodiments, the isolated polynucleotide sequence comprises atleast 80% identity to a compliment of any of polynucleotide sequence ofSEQ ID NOs 1-1690. In some embodiments, the isolated polynucleotidesequence comprises at least 90% identity to a compliment of any ofpolynucleotide sequence of SEQ ID NOs 1-1690. In some embodiments, thepolynucleotide sequence comprises any of a CNV of SEQ ID NOs 1-883. Insome embodiments, the polynucleotide sequence comprises any of a genomicsequence of a gene in Table 3. In some embodiments, the sequencecomprises an RNA sequence transcribed from a genomic sequence of a genein Table 3. In some embodiments, the polynucleotide sequence comprisesany of genetic variation not present in the genome of a subject withouta DD. In some embodiments, the polynucleotide sequence fragmentcomprises a nucleic acid probe. In some embodiments, the nucleic acidprobe is capable of hybridization to a nucleic acid of interest. In someembodiments, the polynucleotide sequence fragment comprises a nucleicacid primer. In some embodiments, the nucleic acid primer is capable ofintiation of extension or amplifying of a nucleic acid of interest.

In one aspect, provided herein is an isolated polypeptide encoded by anRNA sequence transcribed from any of genomic sequence of a gene in Table3.

In one aspect, provided herein is a host cell comprising an expressioncontrol sequence operably linked to a polynucleotide selected from thegroup consisting of any of polynucleotide sequence of a gene in Table 3,or a genetic variant encoded by any one of SEQ ID NOs 1-883. In someembodiments, the expression control sequence is non-native to the hostcell. In some embodiments, the expression control sequence is native tothe host cell.

In one aspect, provided herein is a method for identifying an agenthaving a therapeutic benefit for treatment of a DD, comprising: (a)providing cells comprising at least one genetic variation of SEQ ID NOs1-883, (b) contacting the cells of (a) with a test agent, and (c)analyzing whether the agent has a therapeutic benefit for treatment ofthe cells of (a), thereby identifying agents which have a therapeuticbenefit for treatment of the DD.

In some embodiments, the method further comprises (d) providing cellswhich do not comprise at least one genetic variation of SEQ ID NOs1-883, (e) contacting the cells of (a) and (d) with a test agent, and(f) analyzing whether the agent has a therapeutic benefit for treatmentof the cells of (a) relative to those of (d), thereby identifying agentswhich have a therapeutic benefit for treatment of the DD. In someembodiments, the therapeutic agent has efficacy for the treatment of aDD. In some embodiments a therapeutic agent is identified by the methodresults.

In one aspect, provided herein is a panel of biomarkers for a DDcomprising one or more genes contained in one or more polynucleotidesequences of a gene in Table 3. In some embodiments, the panel comprisestwo or more genes contained in the one or more polynucleotide sequencesselected from the genes in Table 3. In some embodiments, the panelcomprises at least 5, 10, 25, 50, 100 or 200 polynucleotide sequences ofthe genes in Table 3. In some embodiments, at least one of thepolynucleotide sequences is a fragment of the one or more polynucleotidesequences selected from the genes in Table 3. In some embodiments, atleast one of the polynucleotide sequences is a variant of the one ormore polynucleotide sequences selected from the genes in Table 3. Insome embodiments, the panel is selected for analysis of polynucleotideexpression levels for a DD. In some embodiments, the polynucleotideexpression levels are mRNA expression levels. In some embodiments, thepanel is used in the management of patient care for a DD, wherein themanagement includes one or more of risk assessment, early diagnosis,prognosis establishment, patient treatment monitoring, and treatmentefficacy detection. In some embodiments, the panel is used in discoveryof therapeutic intervention of a DD. In some embodiments, at least oneof the biomarkers is attached to substrate. In some embodiments, thesubstrate comprises a plastic, glass, a bead, or a plate. In someembodiments, at least one of the biomarkers is labeled with a detectablelabel. In some embodiments, the panel is an in silico panel.

In one aspect, provided herein is a method for measuring expressionlevels of polynucleotide sequences from biomarkers for a DD in asubject, comprising: (a) selecting a panel of biomarkers comprising twoor more genes contained in one or more polynucleotide sequences selectedfrom the genes in Table 3; (b) isolating cellular RNA from a nucleicacid sample obtained from the subject; (c) synthesizing cDNA from theRNA for each biomarker in the panel using suitable primers; (d)optionally amplifying the cDNA; and (e) quantifying levels of the cDNAfrom the nucleic acid sample. In some embodiments, the step of selectinga panel of biomarkers comprises at least 5, 10, 25, 50, 100 or 200 genescontained in one or more polynucleotide sequences selected from thegenes in Table 3. In some embodiments, the step of quantifying thelevels of cDNA further comprises labeling cDNA. In some embodiments,labeling cDNA comprises labeling with at least one chromophore. In someembodiments, the cDNA levels for the nucleic acid sample are compared toa control cDNA level. In some embodiments, the comparison is used in themanagement of patient care in DD. In some embodiments, the management ofpatient care includes one or more of risk assessment, early diagnosis,establishing prognosis, monitoring patient treatment, and detectingtreatment efficacy. In some embodiments, the comparison is used indiscovery of therapeutic intervention of a DD.

In one aspect, provided herein is a method for measuring expressionlevels of polypeptides comprising: (a) selecting a panel of biomarkerscomprising at least two polypeptides encoded by an RNA sequencetranscribed from a genomic sequence of a gene in Table 3; (b) obtaininga nucleic acid sample; (c) creating an antibody panel for each biomarkerin the panel; (d) using the antibody panel to bind the polypeptides fromthe nucleic acid sample; and (e) quantifying levels of the polypeptidesbound from the nucleic acid sample to the antibody panel. In someembodiments, the polypeptide levels of the nucleic acid sample areincreased or decreased compared to the polypeptide levels of a controlnucleic acid sample. In some embodiments, the subject is treated for aDD patient based on the quantified levels of the polypeptides bound fromthe nucleic acid sample to the antibody panel. In some embodiments, thetreatment of a subject includes one or more of risk assessment, earlydiagnosis, establishing prognosis, monitoring patient treatment, anddetecting treatment efficacy. In some embodiments, the comparison isused in discovery of a therapeutic intervention of a DD.

In one aspect, provided herein is a kit for the determination of a DDcomprising: at least one reagent that is used in analysis of one or morepolynucleotide expression levels for a panel of biomarkers for a DD,wherein the panel comprises two or more genes contained in one or morepolynucleotide sequences selected from the genes in Table 3, andinstructions for using the kit for analyzing the expression levels. Insome embodiments, the one or more polynucleotide expression levelscomprise one or more RNA transcript expression levels. In someembodiments, the one or more RNA transcript expression levels correspondto one or more RNA transcripts of Table 4, or one ore more RNAtranscripts encoded by any of SEQ ID NOs 884-1690. In some embodiments,the at least one reagent comprises at least two sets of suitableprimers. In some embodiments, the at least one reagent comprises areagent for the preparation of cDNA. In some embodiments, the at leastone reagent comprises a reagent that is used for detection andquantization of polynucleotides. In some embodiments, the at least onereagent comprises one or more antibodies wherein the one or moreantibodies detect the one or more polypeptides are translated from oneor more RNA transcripts of Table 4, or the one ore more RNA transcriptsencoded by any of SEQ ID NOs 884-1690. In some embodiments, the at leastone reagent comprises at least one chromophore.

In one aspect, provided herein is a kit for the determination of a DDcomprising: at least one reagent that is used in analysis of polypeptideexpression levels for a panel of biomarkers for DD, wherein the panelcomprises at least two polypeptides expressed from two or more genescontained in one or more polynucleotide sequences selected from thegenes in Table 3; and instructions for using the kit for analyzing theexpression levels. In some embodiments, the reagent is an antibodyreagent that binds a polypeptide selected in the panel. In someembodiments, the kit further comprises a reagent that is used fordetection of a bound polypeptide. In some embodiments, the reagentincludes a second antibody.

In one aspect, provided herein is a method of screening a subject for aDD, the method comprising: (a) assaying a nucleic acid sample obtainedfrom the subject by PCR, aCGH, sequencing, SNP genotyping, orFluorescence in Situ Hybridization to detect sequence information formore than one genetic loci; (b) comparing the sequence information to apanel of nucleic acid biomarkers, wherein the panel comprises at leastone nucleic acid biomarker for each of the more than one genetic loci;and wherein the panel comprises at least 2 low frequency nucleic acidbiomarkers, wherein the low frequency nucleic acid biomarkers occur at afrequency of 0.1% or less in a population of subjects without adiagnosis of the DD; and (c) screening the subject for the presence orabsence of the DD if one or more of the low frequency biomarkers in thepanel are present in the sequence information. In some embodiments, thepanel comprises at least 5, 10, 25, 50, 100 or 200 low frequency nucleicacid biomarkers. In some embodiments, the presence or absence of the DDin the subject is determined with more than 50%, 55%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.8% confidence. In someembodiments, the low frequency biomarkers occur at a frequency of 0.01%or less, 0.001% or less, or 0.0001% or less in a population of subjectswithout a diagnosis of the DD. In some embodiments, the panel of nucleicacid biomarkers comprises at least two genes contained in the one ormore polynucleotide sequences selected from the genes in Table 3. Insome embodiments, the DD is ASD. In some embodiments, the method furthercomprises identifying a therapeutic agent useful for treating the DD. Insome embodiments, the method further comprises administering one or moreof the therapeutic agents to the subject if one or more of the lowfrequency biomarkers in the panel are present in the sequenceinformation.

In one aspect, provided herein is a kit for screening a subject for aDD, the kit comprising at least one reagent for assaying a nucleic acidsample from the subject for information on a panel of nucleic acidbiomarkers, wherein the panel comprises at least 2 low frequencybiomarkers, and wherein the low frequency biomarkers occur at afrequency of 0.1% or less in a population of subjects without adiagnosis of the DD. In some embodiments, a presence or absence of theDD in the subject is determined with more than 50%, 55%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.8% confidence. In someembodiments, the panel comprises at least 5, 10, 25, 50, 100 or 200 lowfrequency nucleic acid biomarkers. In some embodiments, the lowfrequency biomarkers occur at a frequency of 0.01% or less, 0.001% orless, or 0.0001% or less in a population of subjects without a diagnosisof the DD. In some embodiments, the panel of nucleic acid biomarkerscomprises at least two genes contained in the one or more polynucleotidesequences selected from the genes in Table 3. In some embodiments, theat least one reagent comprises at least two sets of suitable primers. Insome embodiments, the at least one reagent comprises a reagent for thepreparation of cDNA. In some embodiments, the at least one reagentcomprises a reagent that is used for detection and quantization ofpolynucleotides. In some embodiments, the at least one reagent comprisesat least one chromophore.

In one aspect, provided herein is a method of generating a panel ofnucleic acid biomarkers comprising: (a) assaying a nucleic acid samplefrom a first population of subjects by PCR, aCGH, sequencing, SNPgenotyping, or Fluorescence in Situ Hybridization for nucleic acidsequence information, wherein the subjects of the first population havea diagnosis of a DD. (b) assaying a nucleic acid sample from a secondpopulation of subjects by PCR, aCGH, sequencing, SNP genotyping, orFluorescence in Situ Hybridization for nucleic acid sequenceinformation, wherein the subjects of the second population are without adiagnosis of a DD; (c) comparing the nucleic acid sequence informationfrom step (a) to that of step (b); (d) determining the frequency of oneor more biomarkers from the comparing step; and (e) generating the panelof a nucleic acid biomarkers, wherein the panel comprises at least 2 lowfrequency biomarkers, and wherein the low frequency biomarkers occur ata frequency of 0.1% or less in a population of subjects without adiagnosis of a DD. In some embodiments, the subjects in the secondpopulation of subjects without a diagnosis of a DD comprise one or moresubjects not suspected of having the DD. In some embodiments, thesubjects in the second population of subjects without a diagnosis of aDD comprise one or more subjects without the DD. In some embodiments,the subjects in the second population of subjects without a diagnosis ofa DD comprise one or more subjects who are asymptomatic for the DD. Insome embodiments, the subjects in the second population of subjectswithout a diagnosis of a DD comprise one or more subjects who havedecreased susceptibility to the DD. In some embodiments, the subjects inthe second population of subjects without a diagnosis of a DD compriseone or more subjects who are unassociated with a treatment, therapeuticregimen, or any combination thereof. In some embodiments, the panelcomprises at least 5, 10, 25, 50, 100 or 200 low frequency nucleic acidbiomarkers. In some embodiments, the low frequency biomarkers occur at afrequency of 0.01% or less, 0.001% or less, or 0.0001% or less in thesecond population of subjects without a diagnosis of a DD In someembodiments, the panel comprises at least two genes contained in the oneor more polynucleotide sequences selected from the genes in Table 3. Insome embodiments, the DD is ASD. In some embodiments, assaying the atleast one nucleic acid sample of the one or more subjects comprisespurifying the at least one nucleic acid sample from the collectedsample. In some embodiments, the method further comprises designing theCGH array to measure one or more genetic variations in Table 1, Table 2,or combinations thereof. In some embodiments, the method furthercomprises providing the CGH array for the measuring of one or moregenetic variations. In some embodiments, assaying at least one nucleicacid sample comprises obtaining the nucleic acid sequence information.In some embodiments, obtaining the nucleic acid information isdetermined by one or more methods selected from the group comprisingPCR, sequencing, Northern blots, FISH, Invader assay, or any combinationthereof. In some embodiments, the at least one genetic variationcomprises one or more point mutations, polymorphisms, single nucleotidepolymorphisms (SNPs), single nucleotide variants (SNVs), translocations,insertions, deletions, amplifications, inversions, microsatellites,interstitial deletions, copy number variations (CNVs), loss ofheterozygosity, or any combination thereof. In some embodiments, the atleast one genetic variation comprises one or more CNVs listed in Table 1or CNV subregions in Table 2. In some embodiments, the genetic variationcomprises one or more CNVs that disrupt, impair, or modulate expressionof one or more genes listed in Table 3. In some embodiments, the atleast one genetic variation comprises one or more CNVs that disrupt,impair, or modulate the expression or function of one or more RNAtranscripts in Table 4, or one ore more RNA transcripts encoded by anyof SEQ ID NOs 884-1690.

In one aspect, provided herein is a method for screening for atherapeutic agent useful for treating a DD, comprising identifying anagent that modulates the function or expression of one or more geneslisted in Table 3 or expression products therefrom. In some embodiments,the expression products comprise one or more RNA transcripts in Table 4,or one ore more RNA transcripts encoded by any of SEQ ID NOs 884-1690.In some embodiments, the expression products comprise one or moreproteins expressed from a gene in Table 3 or encoded by one or more RNAtranscripts in Table 4, or by any of SEQ ID NOs 884-1690. In someembodiments, modulating the function or activity of one or more RNAtranscripts or proteins comprises an increase in expression. In someembodiments, modulating the function or activity of one or more RNAtranscripts or proteins comprises a decrease in expression.

In one aspect, provided herein is a method of treating a subject for aDD, comprising administering one or more agents to modulate the functionof one or more genes listed in Table 3, or expression productstherefrom, thereby treating the DD. In some embodiments, the expressionproducts comprise one or more RNA transcripts in Table 4, or one oremore RNA transcripts encoded by any of SEQ ID NOs 884-1690. In someembodiments, the expression products comprise one or more proteinsexpressed from a gene in Table 3, or encoded by one or more RNAtranscripts in Table 4. In some embodiments, the one or more agents areselected from the group comprising: an antibody, a drug, a combinationof drugs, a compound, a combination of compounds, radiation, a geneticsequence, a combination of genetic sequences, heat, cryogenics, and acombination of two or more of any combination thereof.

In one aspect, provided herein is a kit for screening for a DD in asubject, the kit comprising at least one means for assaying a nucleicacid sample from the subject for the presence of at least one geneticvariation in Table 1 or 2 associated with a DD. In some embodiments, theat least one genetic variation is associated with a disruption oraberration of one or more RNA transcripts in Table 4 or one ore more RNAtranscripts encoded by any of SEQ ID NOs 884-1690. In some embodiments,the at least one genetic variation is associated with a disruption oraberration of one or more proteins expressed from one or more geneslisted in Tables 3, or encoded by one or more RNA transcripts in Table 4or one ore more RNA transcripts encoded by any of SEQ ID NOs 884-1690.In some embodiments, screening the one or more subjects furthercomprises selecting one or more therapies based on the presence orabsence of the one or more genetic variations.

In one aspect, provided herein is a method comprising isolating apoluynucleotide comprising a CNV sequence encoded by any one of SEQ IDNOs 1-883. In some embodiments, assaying the at least one nucleic acidsample of the one or more subjects comprises an analysis of the at leastone collected sample or unamplified nucleic acid sample. In someembodiments, assaying the at least one nucleic acid sample of the one ormore subjects comprises an Invader assay analysis of the at least onecollected sample or unamplified nucleic acid sample. In someembodiments, the method further comprises assaying one or more othergenetic variations in the one or more genes in Table 3, wherein theother genetic variations do not comprise a genetic variation encoded byany one of SEQ ID NOs. 1-883. In some embodiments, the one or more othergenetic variations are shorter in length than one or more of the geneticvariations encoded by any one of SEQ ID NOs. 1-883. In some embodiments,the sequence information of one or more other genetic variations arecompared to a compilation of data comprising frequencies of the othergenetic variations in at least 2 normal human subjects. In someembodiments, the method further comprises determining whether the othergenetic variations are associated with a DD by the comparison. In someembodiments, the assaying comprises analyzing the whole genome or wholeexome from the one or more subjects. In some embodiments, the comparingcomprises determining an odds ratio (OR) value for the one or more othergenetic variations, determining a relative risk value (RR) for the oneor more other genetic variations, or a combination thereof. In someembodiments, determining whether the one or more subjects has a DD or analtered susceptibility to a DD comprises comparing the nucleic acidsequence information, the at least one genetic variation identified inthe one or more subjects, or a combination thereof, to those of one ormore other subjects for enrollment of said subjects or said othersubjects in a clinical trial. In some embodiments, the method furthercomprises detecting one or more genetic variants in an upstream ordownstream region of the one or more genes in Table 3 that results inmodulation of expression of the gene. In some embodiments, the upstreamor downstream region is a gene regulatory sequence. In some embodiments,the method further comprises obtaining sequence information for one ormore of the CNVs encoded by SEQ ID NOs 1-883. In some embodiments, thenucleic acid information further comprises sequence information for oneor more of the CNVs encoded by SEQ ID NOs 1-883. In some embodiments,sequence information for one or more of the CNVs encoded by SEQ ID NOs1-883 comprises nucleic acid information relating to a regulatory regionof a gene in Table 3.

DETAILED DESCRIPTION OF THE DISCLOSURE

The details of one or more inventive embodiments are set forth in theaccompanying drawings, the claims, and in the description herein. Otherfeatures, objects, and advantages of inventive embodiments disclosed andcontemplated herein will be apparent from the description and drawings,and from the claims. As used herein, unless otherwise indicated, thearticle “a” means one or more unless explicitly otherwise provided for.As used herein, unless otherwise indicated, terms such as “contain,”“containing,” “include,” “including,” and the like mean “comprising.” Asused herein, unless otherwise indicated, the term “or” can beconjunctive or disjunctive. As used herein, unless otherwise indicated,any embodiment can be combined with any other embodiment. As usedherein, unless otherwise indicated, some inventive embodiments hereincontemplate numerical ranges. When ranges are present, the rangesinclude the range endpoints. Additionally, every subrange and valuewithin the range is present as if explicitly written out.

Described herein are methods of identifying variations in nucleic acidsand genes associated with one or more developmental conditions.Described herein are methods of screening for determining a subject'ssusceptibility to developing or having, one or more developmentaldisorders, for example Autism Spectrum Disorder (ASD), based onidentification and detection of genetic nucleic acid variations. Alsodescribed herein, are methods and compositions for treating and/orpreventing one or more developmental conditions using a therapeuticmodality. The present disclosure encompasses methods of assessing anindividual for probability of response to a therapeutic agent for adevelopmental disorder, methods for predicting the effectiveness of atherapeutic agent for a developmental disorder, nucleic acids,polypeptides and antibodies and computer-implemented functions. Kits forscreening a sample from a subject to detect or determine susceptibilityto a developmental disorder are also encompassed by the disclosure.

Genetic Variations Associated with Developmental Disorders

Genomic sequences within populations exhibit variability betweenindividuals at many locations in the genome. For example, the humangenome exhibits sequence variations that occur on average every 500 basepairs. Such genetic variations in nucleic acid sequences are commonlyreferred to as polymorphisms or polymorphic sites. As used herein, apolymorphism, e.g. genetic variation, includes a variation in thesequence of a gene in the genome amongst a population, such as allelicvariations and other variations that arise or are observed. Thus, apolymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Thesedifferences can occur in coding and non-coding portions of the genome,and can be manifested or detected as differences in nucleic acidsequences, gene expression, including, for example transcription,processing, translation, transport, protein processing, trafficking, DNAsynthesis; expressed proteins, other gene products or products ofbiochemical pathways or in post-translational modifications and anyother differences manifested amongst members of a population. A singlenucleotide polymorphism (SNP) includes to a polymorphism that arises asthe result of a single base change, such as an insertion, deletion orchange in a base. A polymorphic marker or site is the locus at whichdivergence occurs. Such site can be as small as one base pair (an SNP).Polymorphic markers include, but are not limited to, restrictionfragment length polymorphisms, variable number of tandem repeats(VNTR's), hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats and other repeatingpatterns, simple sequence repeats and insertional elements, such as Alu.Polymorphic forms also are manifested as different mendelian alleles fora gene. Polymorphisms can be observed by differences in proteins,protein modifications, RNA expression modification, DNA and RNAmethylation, regulatory factors that alter gene expression and DNAreplication, and any other manifestation of alterations in genomicnucleic acid or organelle nucleic acids.

In some embodiments, these genetic variations can be found to beassociated with one or more disorders and/or diseases using the methodsdisclosed herein. In some embodiments, these genetic variations can befound to be associated with absence of one or more disorders and/ordiseases (i.e., the one or more variants are protective againstdevelopment of the disorder and/or diseases) using the methods disclosedherein. In some embodiments the one or more disorders and/or diseasescomprise one or more developmental disorders. In some embodiments theone or more developmental disorders comprise one or more PervasiveDevelopmental Disorders (PDD). In some embodiments, the one or more PDDscomprise Autism Spectrum Disorder (ASD), also known as autism. Inanother embodiment, the one or more developmental disorders comprisePervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS). Insome embodiments, PDD can comprise Asperger Syndrome, Rett Syndrome,Fragile X Syndrome and/or Childhood Disintegrative Disorder. In someembodiments genetic variations can be associated with one or more PDDs.In some embodiments genetic variations can be associated with one ormore PDD-NOSs.

Scientific evidence suggests there is a potential for variouscombinations of factors causing ASD, such as multiple genetic variationsthat may cause autism on their own or when combined with exposure to asyet undetermined environmental factors. Timing of exposure during thechild's development, such as before, during, or after birth, may alsoplay a role in the development or final presentation of the disorder. Asmall number of cases can be linked to genetic disorders such as FragileX, Tuberous Sclerosis, and Angelman's Syndrome, as well as exposure toenvironmental agents such as infectious ones (maternal rubella orcytomegalovirus) or chemical ones (thalidomide or valproate) duringpregnancy.

In some embodiments, these genetic variations comprise point mutations,polymorphisms, single nucleotide polymorphisms (SNPs), single nucleotidevariations (SNVs), translocations, insertions, deletions,amplifications, inversions, interstitial deletions, copy numbervariations (CNVs), loss of heterozygosity, or any combination thereof.As genetic variation includes any deletion, insertion or basesubstitution of the genomic DNA of one or more individuals in a firstportion of a total population which thereby results in a difference atthe site of the deletion, insertion or base substitution relative to oneor more individuals in a second portion of the total population. Thus,the term “genetic variation” encompasses “wild type” or the mostfrequently occurring variation, and also includes “mutant,” or the lessfrequently occurring variation.

As used herein, a target molecule that is “associated with” or“correlates with” a particular genetic variation is a molecule that canbe functionally distinguished in its structure, activity, concentration,compartmentalization, degradation, secretion, and the like, as a resultof such genetic variation. In some embodiments polymorphisms (e.g.polymorphic markers, genetic variations, or genetic variants) cancomprise any nucleotide position at which two or more sequences arepossible in a subject population. In some embodiments, each version of anucleotide sequence with respect to the polymorphism can represent aspecific allele, of the polymorphism. In some embodiments, genomic DNAfrom a subject can contain two alleles for any given polymorphic marker,representative of each copy of the marker on each chromosome. In someembodiments, an allele can be a nucleotide sequence of a given locationon a chromosome. Polymorphisms can comprise any number of specificalleles. In some embodiments of the disclosure, a polymorphism can becharacterized by the presence of two or more alleles in a population. Insome embodiments, the polymorphism can be characterized by the presenceof three or more alleles. In some embodiments, the polymorphism can becharacterized by four or more alleles, five or more alleles, six or morealleles, seven or more alleles, nine or more alleles, or ten or morealleles. In some embodiments an allele can be associated with one ormore diseases or disorders, for example, a developmental disorder riskallele can be an allele that is associated with increased or decreasedrisk of developing a developmental disorder. In some embodiments,genetic variations and alleles can be used to associate an inheritedphenotype, for example a developmental disorder, with a responsiblegenotype. In some embodiments, a developmental disorder risk allele canbe a variant allele that is statistically associated with a screening ofone or more developmental disorders. In some embodiments, geneticvariations can be of any measurable frequency in the population, forexample, a frequency higher than 10%, a frequency from 5-10%, afrequency from 1-5%, a frequency from 0.1-1%, or a frequency below 0.1%.As used herein, variant alleles can be alleles that differ from areference allele. As used herein, a variant can be a segment of DNA thatdiffers from the reference DNA, such as a genetic variation. In someembodiments, genetic variations can be used to track the inheritance ofa gene that has not yet been identified, but whose approximate locationis known.

As used herein, a “haplotype” can be information regarding the presenceor absence of one or more genetic markers in a given chromosomal regionin a subject. In some embodiments, a haplotype can be a segment of DNAcharacterized by one or more alleles arranged along the segment, forexample, a haplotype can comprise one member of the pair of alleles foreach genetic variation or locus. In some embodiments, the haplotype cancomprise two or more alleles, three or more alleles, four or morealleles, five or more alleles, or any combination thereof, wherein, eachallele can comprise one or more genetic variations along the segment.

In some embodiments, a genetic variation can be a functional aberrationthat can alter gene function, gene expression, polypeptide expression,polypeptide function, or any combination thereof. In some embodiments, agenetic variation can be a loss-of-function mutation, gain-of-functionmutation, dominant negative mutation, or reversion. In some embodiments,a genetic variation can be part of a gene's coding region or regulatoryregion. Regulatory regions can control gene expression and thuspolypeptide expression. In some embodiments, a regulatory region can bea segment of DNA wherein regulatory polypeptides, for example,transcription or splicing factors, can bind. In some embodiments aregulatory region can be positioned near the gene being regulated, forexample, positions upstream or downstream of the gene being regulated.In some embodiments, a regulatory region (e.g., enhancer element) can beseveral thousands of base pairs upstream or downstream of a gene.

In some embodiments, variants can include changes that affect apolypeptide, such as a change in expression level, sequence, function,localization, binding partners, or any combination thereof. In someembodiments, a genetic variation can be a frameshift mutation, nonsensemutation, missense mutation, neutral mutation, or silent mutation. Forexample, sequence differences, when compared to a reference nucleotidesequence, can include the insertion or deletion of a single nucleotide,or of more than one nucleotide, resulting in a frame shift; the changeof at least one nucleotide, resulting in a change in the encoded aminoacid; the change of at least one nucleotide, resulting in the generationof a premature stop codon; the deletion of several nucleotides,resulting in a deletion of one or more amino acids encoded by thenucleotides; the insertion of one or several nucleotides, such as byunequal recombination or gene conversion, resulting in an interruptionof the coding sequence of a reading frame; duplication of all or a partof a sequence; transposition; or a rearrangement of a nucleotidesequence. Such sequence changes can alter the polypeptide encoded by thenucleic acid, for example, if the change in the nucleic acid sequencecauses a frame shift, the frame shift can result in a change in theencoded amino acids, and/or can result in the generation of a prematurestop codon, causing generation of a truncated polypeptide. In someembodiments, a genetic variation associated with a developmentaldisorder can be a synonymous change in one or more nucleotides, forexample, a change that does not result in a change in the amino acidsequence. Such a polymorphism can, for example, alter splice sites,affect the stability or transport of mRNA, or otherwise affect thetranscription or translation of an encoded polypeptide. In someembodiments, a synonymous mutation can result in the polypeptide producthaving an altered structure due to rare codon usage that impactspolypeptide folding during translation, which in some cases may alterits function and/or drug binding properties if it is a drug target. Insome embodiments, the changes that can alter DNA increase thepossibility that structural changes, such as amplifications ordeletions, occur at the somatic level. A polypeptide encoded by thereference nucleotide sequence can be a reference polypeptide with aparticular reference amino acid sequence, and polypeptides encoded byvariant nucleotide sequences can be variant polypeptides with variantamino acid sequences.

In some embodiments, one or more variant polypeptides can be associatedwith one or more diseases or disorders, such as ASD. In someembodiments, variant polypeptides and changes in expression,localization, and interaction partners thereof, can be used to associatean inherited phenotype, for example, a developmental disorder, with aresponsible genotype. In some embodiments, a developmental disorderassociated variant polypeptide can be statistically associated with adiagnosis, prognosis, or theranosis of one or more developmentaldisorders.

The most common sequence variants comprise base variations at a singlebase position in the genome, and such sequence variants, orpolymorphisms, are commonly called single nucleotide polymorphisms(SNPs) or single nucleotide variants (SNVs). In some embodiments, a SNPrepresents a genetic variant present at greater than or equal to 1%occurrence in a population and in some embodiments a SNP or an SNV canrepresent a genetic variant present at any frequency level in apopulation. A SNP can be a nucleotide sequence variation occurring whena single nucleotide at a location in the genome differs between membersof a species or between paired chromosomes in a subject. SNPs caninclude variants of a single nucleotide, for example, at a givennucleotide position, some subjects can have a ‘G’, while others can havea ‘C’. SNPs can occur in a single mutational event, and therefore therecan be two possible alleles possible at each SNP site; the originalallele and the mutated allele. SNPs that are found to have two differentbases in a single nucleotide position are referred to as biallelic SNPs,those with three are referred to as triallelic, and those with all fourbases represented in the population are quadallelic. In someembodiments, SNPs can be considered neutral. In some embodiments SNPscan affect susceptibility to developmental disorders. SNP polymorphismscan have two alleles, for example, a subject can be homozygous for oneallele of the polymorphism wherein both chromosomal copies of theindividual have the same nucleotide at the SNP location, or a subjectcan be heterozygous wherein the two sister chromosomes of the subjectcontain different nucleotides. The SNP nomenclature as reported hereinis the official Reference SNP (rs) ID identification tag as assigned toeach unique SNP by the National Center for Biotechnological Information(NCBI).

Another genetic variation of the disclosure can be copy numbervariations (CNVs). As used herein, “CNVs” include alterations of the DNAof a genome that results in an abnormal number of copies of one or moresections of DNA. In some embodiments, a CNV comprises a CNV-subregion.As used herein, a “CNV-subregion” includes a continuous nucleotidesequence within a CNV. In some embodiments, the nucleotide sequence of aCNV-subregion can be shorter than the nucleotide sequence of the CNV.CNVs can be inherited or caused by de novo mutation and can beresponsible for a substantial amount of human phenotypic variability,behavioral traits, and disease susceptibility. In some embodiments, CNVsof the current disclosure can be associated with susceptibility to oneor more developmental disorders, for example, Autism Spectrum Disorder.In some embodiments, CNVs can include a single gene or include acontiguous set of genes. In some embodiments, CNVs can be caused bystructural rearrangements of the genome, for example, unbalancedtranslocations, insertions, deletions, amplifications, and interstitialdeletions. In some embodiments, these structural rearrangements occur onone or more chromosomes. Low copy repeats (LCRs), which areregion-specific repeat sequences (also known as segmental duplications),can be susceptible to these structural rearrangements, resulting inCNVs. Factors such as size, orientation, percentage similarity and thedistance between the copies can influence the susceptibility of LCRs togenomic rearrangement. In addition, rearrangements may be mediated bythe presence of high copy number repeats, such as long interspersedelements (LINES) and short interspersed elements (SINEs), often vianon-homologous recombination. For example, chromosomal rearrangementscan arise from non-allelic homologous recombination during meiosis orvia a replication-based mechanism such as fork stalling and templateswitching (FoSTeS) (Zhang F. et al., Nat. Genet., 2009) ormicrohomology-mediated break-induced repair (MMBIR) (Hastings P. J. etal., PLoS Genet., 2009). In some embodiments, CNVs are referred to asstructural variants, which are a broader class of variant that alsoincludes copy number neutral alterations such as inversions and balancedtranslocations. In some embodiments, CNVs are referred to as structuralvariants. In some embodiments, structural variants can be a broaderclass of variant that can also include copy number neutral alterationssuch as inversions and balanced translocations.

CNVs can account for genetic variation affecting a substantialproportion of the human genome, for example, known CNVs can cover over15% of the human genome sequence (Estivill, X and Armengol, L., PLoSGenetics, 2007). CNVs can affect gene expression, phenotypic variationand adaptation by disrupting or impairing gene dosage, and can causedisease, for example, microdeletion and microduplication disorders, andcan confer susceptibility to diseases and disorders. Updated informationabout the location, type, and size of known CNVs can be found in one ormore databases, for example, the Database of Genomic Variants, whichcurrently contains data for over 100,000 CNVs (as of September, 2013).

Other types of sequence variants can be found in the human genome andcan be associated with a disease or disorder, including but not limitedto, microsatellites. Microsatellite markers are stable, polymorphic,easily analyzed, and can occur regularly throughout the genome, makingthem especially suitable for genetic analysis. A polymorphicmicrosatellite can comprise multiple small repeats of bases, forexample, CA repeats, at a particular site wherein the number of repeatlengths varies in a population. In some embodiments, microsatellites,for example, variable number of tandem repeats (VNTRs), can be shortsegments of DNA that have one or more repeated sequences, for example,about 2 to 5 nucleotides long, that can occur in non-coding DNA. In someembodiments, changes in microsatellites can occur during geneticrecombination of sexual reproduction, increasing or decreasing thenumber of repeats found at an allele, or changing allele length.

Developmental Disorders

Developmental disorders are disorders that occur at some stage in achild's development, often retarding the development, includingpsychological or physical disorders. In some embodiments, they can bedistinguished into specific developmental disorders including PervasiveDevelopmental Disorders (PDDs) and Pervasive Developmental Disorder—NotOtherwise Specified (PDD-NOS). In a preferred embodiment of the presentdisclosure, a PDD can comprise Autism Spectrum Disorder (ASD).Generally, symptoms that may be present to some degree in a subject ofthe present disclosure with a PDD can include difficulty with verbalcommunication, including problems using and understanding language,difficulty with non-verbal communication, such as gestures and facialexpressions such as smiling, difficulty with social interaction,including relating to people and to his or her surroundings, unusualways of playing with toys and other objects, difficulty adjusting tochanges in routine or familiar surroundings, repetitive body movementsor patterns of behavior, such as hand flapping, spinning, and headbanging, changing response to sound, temper tantrums, difficultysleeping, aggressive behavior, and/or fearfulness or anxiety. ASD can bedefined by a certain set of behaviors that can range from the very mildto the severe. Possible indicators of Autism Spectrum Disorders includea subject whom does not babble, point, or make meaningful gestures by 1year of age; does not speak one word by 16 months, does not combine twowords by 2 years, does not respond to their name, and/or loses languageor social skills.

As described herein, Pervasive Developmental Disorders—Not OtherwiseSpecified (PDD-NOS) can comprise Asperger Syndrome, Rett Syndrome,Fragile X Syndrome, and/or Childhood Disintegrative Disorder. In someembodiments a screening of PDD-NOS can be a screening of being on theautism spectrum, but not falling within any of the existing specificcategories of autism. PDD-NOS is a pervasive developmental disorder(PDD)/autism spectrum disorder (ASD) and is often referred to asatypical autism.

Subjects

A “subject”, as used herein, can be an individual of any age or sex fromwhom a sample containing nucleotides is obtained for analysis by one ormore methods described herein so as to obtain nucleic acid information,for example, a male or female adult, child, newborn, or fetus. In someembodiments, a subject can be any target of therapeutic administration.In some embodiments, a subject can be a test subject or a referencesubject. In some embodiments, a subject can be associated with acondition or disease or disorder, asymptomatic or symptomatic, haveincreased or decreased susceptibility to a disease or disorder, beassociated or unassociated with a treatment or treatment regimen, or anycombination thereof.

As used herein, a “cohort” can represent an ethnic group, a patientgroup, a particular age group, a group not associated with a particulardisease or disorder, a group associated with a particular disease ordisorder, a group of asymptomatic subjects, a group of symptomaticsubjects, or a group or subgroup of subjects associated with aparticular response to a treatment regimen or clinical trial. In someembodiments, a patient can be a subject afflicted with a disease ordisorder. In some embodiments, a patient can be a subject not afflictedwith a disease or disorder and is considered apparently healthy, or anormal or control subject. In some embodiments, a subject can be a testsubject, a patient or a candidate for a therapeutic, wherein genomic DNAfrom the subject, patient, or candidate is obtained for analysis by oneor more methods of the present disclosure herein, so as to obtaingenetic variation information of the subject, patient or candidate.

In some embodiments, the nucleic acid sample can be obtained prenatallyfrom a fetus or embryo or from the mother, for example, from fetal orembryonic cells in the maternal circulation. In some embodiments, thenucleic acid sample can be obtained with the assistance of a health careprovider, for example, to draw blood. In some embodiments, the nucleicacid sample can be obtained without the assistance of a health careprovider, for example, where the nucleic acid sample is obtainednon-invasively, such as a saliva sample, or a sample comprising buccalcells that is obtained using a buccal swab or brush, or a mouthwashsample.

The present disclosure also provides methods for assessing geneticvariations in subjects who are members of a target population. Such atarget population is in some embodiments a population or group ofsubjects at risk of developing the disease, based on, for example, othergenetic factors, biomarkers, biophysical parameters, diagnostic testingsuch as magnetic resonance imaging (MRI), family history of adevelopmental disorder, previous screening or medical history, or anycombination thereof.

Although ASD is known to affect children to a higher extent than adults,subjects of all ages are contemplated in the present disclosure. In someembodiments subjects can be from specific age subgroups, such as thoseover the age of 1, over the age of 2, over the age of 3, over the age of4, over the age of 5, over the age of 6, over the age of 7, over the ageof 8, over the age of 9, over the age of 10, over the age of 15, overthe age of 20, over the age of 25, over the age of 30, over the age of35, over the age of 40, over the age of 45, over the age of 50, over theage of 55, over the age of 60, over the age of 65, over the age of 70,over the age of 75, over the age of 80, or over the age of 85. Otherembodiments of the disclosure pertain to other age groups, such assubjects aged less than 85, such as less than age 80, less than age 75,less than age 70, less than age 65, less than age 60, less than age 55,less than age 50, less than age 45, less than age 40, less than age 35,less than age 30, less than age 25, less than age 20, less than age 15,less than age 10, less than age 9, less than age 8, less than age 7,less than age 6, less than age 5, less than age 4, less than age 3, lessthan age 2, or less than age 1. Other embodiments relate to subjectswith age at onset of the disease in any of particular age or age rangesdefined by the numerical values described in the above or othernumerical values bridging these numbers. It is also contemplated that arange of ages can be relevant in certain embodiments, such as age atonset at more than age 15 but less than age 20. Other age ranges arehowever also contemplated, including all age ranges bracketed by the agevalues listed in the above.

The genetic variations of the present disclosure found to be associatedwith a developmental disorder can show similar association in otherhuman populations. Particular embodiments comprising subject humanpopulations are thus also contemplated and within the scope of thedisclosure. Such embodiments relate to human subjects that are from oneor more human populations including, but not limited to, Caucasian,Ashkenazi Jewish, Sephardi Jewish, European, American, Eurasian, Asian,Central/South Asian, East Asian, Middle Eastern, African, Hispanic, andOceanic populations. European populations include, but are not limitedto, Swedish, Norwegian, Finnish, Russian, Danish, Icelandic, Irish,Kelt, English, Scottish, Dutch, Belgian, French, German, Spanish,Portuguese, Italian, Polish, Bulgarian, Slavic, Serbian, Bosnian, Czech,Greek and Turkish populations. The ethnic contribution in subjects canalso be determined by genetic analysis, for example, genetic analysis ofancestry can be carried out using unlinked microsatellite markers orsingle nucleotide polymorphisms (SNPs) such as those set out in Smith etal. (Smith M. W. et al., 2004, Am. J. Hum. Genet. 74:1001).

It is also well known to the person skilled in the art that certaingenetic variations have different population frequencies in differentpopulations, or are polymorphic in one population but not in another. Aperson skilled in the art can however apply the methods available and asthought herein to practice the present disclosure in any given humanpopulation. This can include assessment of genetic variations of thepresent disclosure, so as to identify those markers that give strongestassociation within the specific population. Thus, the at-risk variantsof the present disclosure can reside on different haplotype backgroundand in different frequencies in various human populations.

Samples

Samples that are suitable for use in the methods described herein can benucleic acid samples from a subject. A “nucleic acid sample” as usedherein can include RNA, DNA, polypeptides, or a combination thereof.Nucleic acids and polypeptides can be extracted from one or more nucleicacid samples including but not limited to, blood, saliva, urine, mucosalscrapings of the lining of the mouth, expectorant, serum, tears, skin,tissue, or hair. A nucleic acid sample can be assayed for nucleic acidinformation. “Nucleic acid information,” as used herein, includes anucleic acid sequence itself, the presence/absence of genetic variationin the nucleic acid sequence, a physical property which varies dependingon the nucleic acid sequence (for example, Tm), and the amount of thenucleic acid (for example, number of mRNA copies). A “nucleic acid”means any one of DNA, RNA, DNA including artificial nucleotides, or RNAincluding artificial nucleotides. As used herein, a “purified nucleicacid” includes cDNAs, fragments of genomic nucleic acids, nucleic acidsproduced polymerase chain reaction (PCR), nucleic acids formed byrestriction enzyme treatment of genomic nucleic acids, recombinantnucleic acids, and chemically synthesized nucleic acid molecules. A“recombinant” nucleic acid molecule includes a nucleic acid moleculemade by an artificial combination of two otherwise separated segments ofsequence, e.g., by chemical synthesis or by the manipulation of isolatedsegments of nucleic acids by genetic engineering techniques. As usedherein, a “polypeptide” includes proteins, fragments of proteins, andpeptides, whether isolated from natural sources, produced by recombinanttechniques, or chemically synthesized. A polypeptide may have one ormore modifications, such as a post-translational modification (e.g.,glycosylation, etc.) or any other modification (e.g., pegylation, etc.).The polypeptide may contain one or more non-naturally-occurring aminoacids (e.g., such as an amino acid with a side chain modification).

In some embodiments, the nucleic acid sample can comprise cells ortissue, for example, cell lines. Exemplary cell types from which nucleicacids can be obtained using the methods described herein and include butare not limited to, a blood cell; such as a B lymphocyte, T lymphocyte,leukocyte, erythrocyte, macrophage, or neutrophil; a muscle cell such asa skeletal cell, smooth muscle cell or cardiac muscle cell; a germ cell,such as a sperm or egg; an epithelial cell; a connective tissue cell,such as an adipocyte, chondrocyte; fibroblast or osteoblast; a neuron;an astrocyte; a stromal cell; an organ specific cell, such as a kidneycell, pancreatic cell, liver cell, or a keratinocyte; a stem cell; orany cell that develops there from. A cell from which nucleic acids canbe obtained can be a blood cell or a particular type of blood cellincluding, for example, a hematopoietic stem cell or a cell that arisesfrom a hematopoietic stem cell such as a red blood cell, B lymphocyte, Tlymphocyte, natural killer cell, neutrophil, basophil, eosinophil,monocyte, macrophage, or platelet. Generally any type of stem cell canbe used including, without limitation, an embryonic stem cell, adultstem cell, or pluripotent stem cell.

In some embodiments, a nucleic acid sample can be processed for RNA orDNA isolation, for example, RNA or DNA in a cell or tissue sample can beseparated from other components of the nucleic acid sample. Cells can beharvested from a nucleic acid sample using standard techniques known inthe art, for example, by centrifuging a cell sample and resuspending thepelleted cells, for example, in a buffered solution, for example,phosphate-buffered saline (PBS). In some embodiments, after centrifugingthe cell suspension to obtain a cell pellet, the cells can be lysed toextract DNA. In some embodiments, the nucleic acid sample can beconcentrated and/or purified to isolate DNA. All nucleic acid samplesobtained from a subject, including those subjected to any sort offurther processing, are considered to be obtained from the subject. Insome embodiments, standard techniques and kits known in the art can beused to extract RNA or DNA from a nucleic acid sample, including, forexample, phenol extraction, a QIAamp® Tissue Kit (Qiagen, Chatsworth,Calif.), a Wizard® Genomic DNA purification kit (Promega), or a QiagenAutopure method using Puregene chemistry, which can enable purificationof highly stable DNA well-suited for archiving.

In some embodiments, determining the identity of an allele ordetermining copy number can, but need not, include obtaining a nucleicacid sample comprising RNA and/or DNA from a subject, and/or assessingthe identity, copy number, presence or absence of one or more geneticvariations and their chromosomal locations within the genomic DNA (i.e.,subject's genome) derived from the nucleic acid sample.

The individual or organization that performs the determination need notactually carry out the physical analysis of a nucleic acid sample from asubject. In some embodiments, the methods can include using informationobtained by analysis of the nucleic acid sample by a third party. Insome embodiments, the methods can include steps that occur at more thanone site. For example, a nucleic acid sample can be obtained from asubject at a first site, such as at a health care provider or at thesubject's home in the case of a self-testing kit. The nucleic acidsample can be analyzed at the same or a second site, for example, at alaboratory or other testing facility.

Methods of Screening

As used herein, screening a subject comprises diagnosing or determining,theranosing, or determining the susceptibility to developing(prognosing) a developmental disorder, for example, ASD. In particularembodiments, the disclosure is a method of determining a presence of, ora susceptibility to, a developmental disorder, by detecting at least onegenetic variation in a sample from a subject as described herein. Insome embodiments, detection of particular alleles, markers, variations,or haplotypes is indicative of a presence or susceptibility to adevelopmental disorder. Although there can be many concerns aboutscreening a subject with an ASD, the earlier the screening of ASD ismade, the earlier needed interventions can begin. Evidence over the last15 years indicates that intensive early intervention in optimaleducational settings for at least 2 years during the preschool yearsresults in improved outcomes in most young children with ASD. Inevaluating a child, clinicians rely on behavioral characteristics tomake a diagnosis, prognosis, or theranosis. Some of the characteristicbehaviors of ASD may be apparent in the first few months of a child'slife, or they may appear at any time during the early years. For thescreening problems in at least one of the areas of communication,socialization, or restricted behavior must be present before the age of3. The screening requires a two-stage process. The first stage involvesdevelopmental screening during “well-child” check-ups; the second stageentails a comprehensive evaluation by a multidisciplinary team. A “wellchild” check-up should include a developmental screening test. Severalscreening instruments have been developed to quickly gather informationabout a child's social and communicative development within medicalsettings. Among them are the Checklist of Autism in Toddlers (CHAT), themodified Checklist for Autism in Toddlers (M-CHAT), the Screening Toolfor Autism in Two-Year-Olds (STAT), and the Social CommunicationQuestionnaire (SCQ) for children 4 years of age and older. Somescreening instruments rely solely on parent responses to aquestionnaire, and some rely on a combination of parent report andobservation. Key items on these instruments that appear to differentiatechildren with autism from other groups before the age of 2 includepointing and pretend play. Screening instruments do not provideindividual diagnosis, prognosis, or theranosis, but serve to assess theneed for referral for possible screening of ASD. These screening methodsmay not identify children with mild ASD, such as those withhigh-functioning autism or Asperger syndrome. The second stage ofscreening must be comprehensive in order to accurately rule in or ruleout an ASD or other developmental problem. This evaluation may be doneby a multidisciplinary team that includes a psychologist, a neurologist,a psychiatrist, a speech therapist, or other professionals who screenchildren with ASD. Because ASDs are complex disorders and may involveother developmental or genetic problems, a comprehensive evaluationshould entail developmental and genetic assessment, along with in-depthcognitive and language testing. In addition, measures developedspecifically for screening autism are often used. These include theAutism Diagnosis Interview-Revised (ADI-R) and the Autism DiagnosticObservation Schedule (ADOS-G). The ADI-R is a structured interview thatcontains over 100 items and is conducted with a caregiver. It consistsof four main factors including the child's communication, socialinteraction, repetitive behaviors, and age-of-onset symptoms. The ADOS-Gis an observational measure used to “press” for socio-communicativebehaviors that are often delayed, abnormal, or absent in children withASD. Still another instrument often used by professionals is theChildhood Autism Rating Scale (CARS). It can aid in evaluating thechild's body movements, adaptation to change, listening response, verbalcommunication, and relationship to people. It is suitable for use withchildren over 2 years of age. The examiner observes the child and alsoobtains relevant information from the parents. The child's behavior israted on a scale based on deviation from the typical behavior ofchildren of the same age. Two other tests that can be used to assess anychild with a developmental delay are a formal audiologic hearingevaluation and a lead screening. Although some hearing loss can co-occurwith ASD, some children with ASD may be incorrectly thought to have sucha loss. In addition, if the child has suffered from an ear infection,transient hearing loss can occur. Lead screening is essential forchildren who remain for a long period of time in the oral-motor stage inwhich they put any and everything into their mouths. Children with anautistic disorder usually have elevated blood lead levels. Customarily,an expert screening team has the responsibility of thoroughly evaluatingthe child, assessing the child's unique strengths and weaknesses, anddetermining a formal screen. The team will then meet with the parents toexplain the results of the evaluation.

PDD-NOS is typically screened by psychologists and PediatricNeurologists. No singular specific test can be administered to determinewhether or not a child is on the spectrum. Screening can be made throughobservations, questionnaires, and tests. A parent will usually initiatethe quest into the screening with questions for their child'spediatrician about their child's development after noticingabnormalities. From there, doctors will ask questions to gauge thechild's development in comparison to age-appropriate milestones. Onetest that measures this is the Modified Checklist of Autism in Toddlers(MCHAT). This is a list of questions whose answers will determinewhether or not the child should be referred to a specialist such as adevelopmental pediatrician, a neurologist, a psychiatrist, or apsychologist. Another checklist, the DSM-IV is a series ofcharacteristics and criteria to qualify for an autism diagnosis. BecausePDD-NOS is a spectrum disorder, not every child shows the same signs.The two main characteristics of the disorder are difficulties withsocial interaction skills and communication. Signs are often visible inbabies but a diagnosis is usually not made until around age 4. Eventhough PDD-NOS is considered milder than typical autism, this is notalways true. While some characteristics may be milder, others may bemore severe. Once a child with PDD-NOS enters school, he or she willoften be very eager to interact with classmates, but may act sociallydifferent to peers and be unable to make genuine connections. As theyage, the closest connections they make are typically with their parents.Children with PDD-NOS have difficulty reading facial expressions andrelating to feelings of others. They may not know how to respond whensomeone is laughing or crying. Literal thinking is also characteristicof PDD-NOS. They will most likely have difficulty understandingfigurative speech and sarcasm. Inhibited communication skills are a signof PDD-NOS that begins immediately after birth. As an infant, they willnot babble, and as they age, they do not speak when age appropriate.Once verbal communication begins, their vocabulary is often limited.Some characteristics of language-based patterns are: repetitive or rigidlanguage, narrow interests, uneven language development, and poornonverbal communication. A very common characteristic of PDD-NOS issevere difficulty grasping the difference between pronouns, particularlybetween “you” and “me” when conversing. During the last few years,screening instruments have been devised to screen for Asperger syndromeand higher functioning autism. The Autism Spectrum ScreeningQuestionnaire (ASSQ), the Australian Scale for Asperger's Syndrome, andthe most recent, the Childhood Asperger Syndrome Test (CAST), are someof the instruments that are reliable for identification of school-agechildren with Asperger syndrome or higher functioning autism. Thesetools concentrate on social and behavioral impairments in childrenwithout significant language delay. If, following the screening processor during a routine “well child” check-up, a subject's doctor sees anyof the possible indicators of ASD, further evaluation is indicated.

While means for screening ASDs exist, many times symptoms go unnoticeduntil late in childhood or symptoms are so minor they are leftunnoticed. Thus there exists a need for an improved ASD screening test.Described herein are methods of screening an individual for one or moredevelopmental disorders, including but not limited to, determining theidentity and location of genetic variations, such as variations innucleotide sequence and copy number, and the presence or absence ofalleles or genotypes in one or more samples from one or more subjectsusing any of the methods described herein. In some embodiments,determining an association to having or developing a developmentaldisorder can be performed by detecting particular variations that appearmore frequently in test subjects compared to reference subjects andanalyzing the molecular and physiological pathways these variations canaffect.

Within any given population, there can be an absolute susceptibility ofdeveloping a disease or trait, defined as the chance of a persondeveloping the specific disease or trait over a specified time-period.Susceptibility (e.g. being at-risk) is typically measured by looking atvery large numbers of people, rather than at a particular individual. Asdescribed herein, certain copy number variations (genetic variations)are found to be useful for susceptibility assessment of a developmentaldisorder. Susceptibility assessment can involve detecting particulargenetic variations in the genome of individuals undergoing assessment.Particular genetic variations are found more frequently in individualswith a developmental disorder, than in individuals without adevelopmental disorder. Therefore, these genetic variations havepredictive value for detecting a developmental disorder, or asusceptibility to a developmental disorder, in an individual. Withoutintending to be limited by theory, it is believed that the geneticvariations described herein to be associated with susceptibility of adevelopmental disorder represent functional variants predisposing to thedisease. In some embodiments, a genetic variation can confer asusceptibility of the condition, for example carriers of the geneticvariation are at a different risk of the condition than non-carriers. Insome embodiments, the presence of a genetic variation is indicative ofincreased susceptibility to a developmental disorder, such as AutismSpectrum Disorder.

In some embodiments, screening can be performed using any of the methodsdisclosed, alone or in combination. In some embodiments, screening canbe performed using Polymerase Chain Reaction (PCR). In some embodimentsscreening can be performed using Array Comparative Genomic Hybridization(aCGH) to detect CNVs. In another preferred embodiment screening can beperformed using exome sequencing to detect SNVs, indels, and in somecases CNVs using appropriate analysis algorithms. In another preferredembodiment screening is performed using high-throughput (also known asnext generation) whole genome sequencing methods and appropriatealgorithms to detect all or nearly all genetic variations present in agenomic DNA sample. In some embodiments, the genetic variationinformation as it relates to the current disclosure can be used inconjunction with any of the above mentioned symptomatic screening teststo screen a subject for ASD, for example, using a combination of aCGHand a childhood screening test, such as the Checklist of Autism inToddlers (CHAT).

In some embodiments, information from any of the above screening methods(e.g. specific symptoms, scoring matrix, or genetic variation data) canbe used to define a subject as a test subject or reference subject. Insome embodiments, information from any of the above screening methodscan be used to associate a subject with a test or reference population,for example, a subject in a population. In the present study, forexample, all the probands in Table 1 met the criteria for autism on oneor both of the screening measures including the Autism DiagnosticInterview-Revised (ADI-R) training and the Autism Diagnostic ObservationSchedule (ADOS) training.

In one embodiment, an association with a developmental disorder can bedetermined by the statistical likelihood of the presence of a geneticvariation in a subject with a developmental disorder, for example, anunrelated individual or a first or second-degree relation of thesubject. In some embodiments, an association with a developmentaldisorder can be determined by determining the statistical likelihood ofthe absence of a genetic variation in an unaffected reference subject,for example, an unrelated individual or a first or second-degreerelation of the subject. The methods described herein can includeobtaining and analyzing a nucleic acid sample from one or more suitablereference subjects.

In the present context, the term screening comprises diagnosis,prognosis, and theranosis. Screening can refer to any availablescreening method, including those mentioned herein. As used herein,susceptibility can be proneness of a subject towards the development ofa developmental condition, or towards being less able to resist aparticular developmental condition than one or more control subjects. Insome embodiments, susceptibility can encompass increased susceptibility.For example, particular nucleic acid variations of the disclosure asdescribed herein can be characteristic of increased susceptibility todevelopment of a developmental disorder. In some embodiments, particularnucleic acid variations can confer decreased susceptibility, for exampleparticular nucleic variations of the disclosure as described herein canbe characteristic of decreased susceptibility to development of adevelopmental disorder.

As described herein, a genetic variation predictive of susceptibility toor presence of a developmental disorder can be one where the particulargenetic variation is more frequently present in a group of subjects withthe condition (affected), compared to the frequency of its presence in areference group (control), such that the presence of the geneticvariation is indicative of susceptibility to or presence of thedevelopmental disorder. In some embodiments, the reference group can bea population nucleic acid sample, for example, a random nucleic acidsample from the general population or a mixture of two or more nucleicacid samples from a population. In some embodiments, disease-freecontrols can be characterized by the absence of one or more specificdisease-associated symptoms, for example, individuals who have notexperienced symptoms associated with a developmental disorder. In someembodiments, the disease-free control group is characterized by theabsence of one or more disease-specific risk factors, for example, atleast one genetic and/or environmental risk factor. In some embodiments,a reference sequence can be referred to for a particular site of geneticvariation. In some embodiments, a reference allele can be a wild-typeallele and can be chosen as either the first sequenced allele or as theallele from a control individual. In some embodiments, one or morereference subjects can be characteristically matched with one or moreaffected subjects, for example, with matched aged, gender or ethnicity.

A person skilled in the art can appreciate that for genetic variationswith two or more alleles present in the population being studied, andwherein one allele can found in increased frequency in a group ofindividuals with a developmental disorder in the population, comparedwith controls, the other allele of the marker can be found in decreasedfrequency in the group of individuals with the trait or disease,compared with controls. In such a case, one allele of the marker, forexample, the allele found in increased frequency in individuals with adevelopmental disorder, can be the at-risk allele, while the otherallele(s) can be a neutral or protective allele.

A genetic variant associated with a developmental disorder can be usedto predict the susceptibility of the disease for a given genotype. Forany genetic variation, there can be one or more possible genotypes, forexample, homozygote for the at-risk variant (e.g., in autosomalrecessive disorders), heterozygote, and non-carrier of the at-riskvariant. Autosomal recessive disorders can also result from twodistinict genetic variants impacting the same gene such that theindividual is a compound heterozygote (e.g., the maternal allelecontains a different mutation than the paternal allele). Compoundheterozygosity may result from two different SNVs, two different CNVs,an SNV and a CNV, or any combination of two different genetic variantsbut each present on a different allele for the gene. For X-linked genes,males who possess one copy of a variant-containing gene may be affected,while carrier females, who also possess a wild-type gene, may remainunaffected. In some embodiments, susceptibility associated with variantsat multiple loci can be used to estimate overall susceptibility. Formultiple genetic variants, there can be k (k=3{circumflex over( )}n*2{circumflex over ( )}P) possible genotypes; wherein n can be thenumber of autosomal loci and p can be the number of gonosomal (sexchromosomal) loci. Overall susceptibility assessment calculations canassume that the relative susceptibilities of different genetic variantsmultiply, for example, the overall susceptibility associated with aparticular genotype combination can be the product of the susceptibilityvalues for the genotype at each locus. If the susceptibility presentedis the relative susceptibility for a person, or a specific genotype fora person, compared to a reference population, then the combinedsusceptibility can be the product of the locus specific susceptibilityvalues and can correspond to an overall susceptibility estimate comparedwith a population. If the susceptibility for a person is based on acomparison to non-carriers of the at-risk allele, then the combinedsusceptibility can correspond to an estimate that compares the personwith a given combination of genotypes at all loci to a group ofindividuals who do not carry at-risk variants at any of those loci. Thegroup of non-carriers of any at-risk variant can have the lowestestimated susceptibility and can have a combined susceptibility,compared with itself, for example, non-carriers, of 1.0, but can have anoverall susceptibility, compared with the population, of less than 1.0.

Overall risk for multiple risk variants can be performed using standardmethodology. Genetic variations described herein can form the basis ofrisk analysis that combines other genetic variations known to increaserisk of a developmental disorder, or other genetic risk variants for adevelopmental disorder. In certain embodiments of the disclosure, aplurality of variants (genetic variations, variant alleles, and/orhaplotypes) can be used for overall risk assessment. These variants arein some embodiments selected from the genetic variations as disclosedherein. Other embodiments include the use of the variants of the presentdisclosure in combination with other variants known to be useful forscreening a susceptibility to a developmental disorder. In suchembodiments, the genotype status of a plurality of genetic variations,markers and/or haplotypes is determined in an individual, and the statusof the individual compared with the population frequency of theassociated variants, or the frequency of the variants in clinicallyhealthy subjects, such as age-matched and sex-matched subjects.

Methods such as the use of available algorithms and software can be usedto identify, or call, significant genetic variations, including but notlimited to, algorithms of DNA Analytics or DNAcopy, iPattern and/orQuantiSNP. In some embodiments, a threshold logratio value can be usedto determine losses and gains. For example, using DNA Analytics, a log₂ratio cutoff of >0.25 and <0.25 to classify CNV gains and lossesrespectively can be used. As a further example, using DNAcopy, a log₂ratio cutoff of >0.35 and <0.35 to classify CNV gains and lossesrespectively can be used. For example, an Aberration Detection Module 2(ADM2) algorithm, such as that of DNA Analytics 4.0.85 can be used toidentify, or call, significant genetic variations. In some embodiments,two or more algorithms can be used to identify, or call, significantgenetic variations. For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morealgorithms can be used to identify, or call, significant geneticvariations. In some embodiments, significant genetic variations can beCNVs.

CNVs detected by 2 or more algorithms can be defined as stringent andcan be utilized for further analyses. In some embodiments, theinformation and calls from two or more of the methods described hereincan be compared to each other to identify significant genetic variationsmore or less stringently. For example, CNV calls generated by two ormore of DNA Analytics, Aberration Detection Module 2 (ADM2) algorithms,and DNAcopy algorithms can be defined as stringent CNVs. In someembodiments significant or stringent genetic variations can be tagged asidentified or called if it can be found to have a minimal reciprocaloverlap to a genetic variation detected by one or more platforms and/ormethods described herein. For example, a minimum of 50% reciprocaloverlap can be used to tag the CNVs as identified or called. Forexample, significant or stringent genetic variations can be tagged asidentified or called if it can be found to have a reciprocal overlap ofmore than about 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, 99%,or equal to 100%, to a genetic variation detected by one or moreplatforms and/or methods described herein. For example, significant orstringent genetic variations can be tagged as identified or called if itcan be found to have a reciprocal overlap of more than about 50%reciprocal overlap to a genetic variation detected by one or moreplatforms and/or methods described herein. In another embodiment,genetic variations can be detected from the log 2 ratio valuescalculated for individual probes present on an aCGH microarray via astatistical comparison of the probe's log 2 ratio value in a cohort ofsubjects with the disease or developmental disorder (e.g., autism) tothe probe's log 2 ratio value in a cohort of subjects without thedisease or developmental disorder (e.g., autism).

In some embodiments, a threshold log ratio value can be used todetermine losses and gains. A log ratio value can be any log ratiovalue; for example, a log ratio value can be a log 2 ratio or a log 10ratio. In some embodiments, a CNV segment whose median log 2 ratio isless than or equal to a log 2 ratio threshold value can be classified asa loss. For example, any segment whose median log 2 ratio is less thanor equal to −0.1, −0.11, −0.12, −0.13, −0.14, −0.15, −0.16, −0.17,−0.18, −0.19, −0.2, −0.21, −0.22, −0.23, −0.24, −0.25, −0.26, −0.27,−0.28, −0.29, −0.3, −0.31, −0.32, −0.33, −0.34, −0.35, −0.36, −0.37,−0.38, −0.39, −0.4, −0.41, −0.42, −0.43, −0.44, −0.45, −0.46, −0.47,−0.48, −0.49, −0.5, −0.55, −0.6, −0.65, −0.7, −0.75, −0.8, −0.85, −0.9,−0.95, −1, −1.1, −1.2, −1.3, −1.4, −1.5, −1.6, −1.7, −1.8, −1.9, −2,−2.1, −2.2, −2.3, −2.4, −2.5, −2.6, −2.7, −2.8, −2.9, −3, −3.1, −3.2,−3.3, −3.4, −3.5, −3.6, −3.7, −3.8, −3.9, −4, −4.1, −4.2, −4.3, −4.4,−4.5, −4.6, −4.7, −4.8, −4.9, −5, −5.5, −6, −6.5, −7, −7.5, −8, −8.5,−9, −9.5, −10, −11, −12, −13, −14, −15, −16, −17, −18, −19, −20 or less,can be classified as a loss.

In some embodiments, one algorithm can be used to call or identifysignificant genetic variations, wherein any segment whose median log 2ratio was less than or equal to −0.1, −0.11, −0.12, −0.13, −0.14, −0.15,−0.16, −0.17, −0.18, −0.19, −0.2, −0.21, −0.22, −0.23, −0.24, −0.25,−0.26, −0.27, −0.28, −0.29, −0.3, −0.31, −0.32, −0.33, −0.34, −0.35,−0.36, −0.37, −0.38, −0.39, −0.4, −0.41, −0.42, −0.43, −0.44, −0.45,−0.46, −0.47, −0.48, −0.49, −0.5, −0.55, −0.6, −0.65, −0.7, −0.75, −0.8,−0.85, −0.9, −0.95, −1, −1.1, −1.2, −1.3, −1.4, −1.5, −1.6, −1.7, −1.8,−1.9, −2, −2.1, −2.2, −2.3, −2.4, −2.5, −2.6, −2.7, −2.8, −2.9, −3,−3.1, −3.2, −3.3, −3.4, −3.5, −3.6, −3.7, −3.8, −3.9, −4, −4.1, −4.2,−4.3, −4.4, −4.5, −4.6, −4.7, −4.8, −4.9, −5, −5.5, −6, −6.5, −7, −7.5,−8, −8.5, −9, −9.5, −10, −11, −12, −13, −14, −15, −16, −17, −18, −19,−20 or less, can be classified as a loss. For example, any CNV segmentwhose median log 2 ratio is less than −0.35 as determined by DNAcopy canbe classified as a loss. For example, losses can be determined accordingto a threshold log 2 ratio, which can be set at −0.35.

In some embodiments, two algorithms can be used to call or identifysignificant genetic variations, wherein any segment whose median log 2ratio is less than or equal to −0.1, −0.11, −0.12, −0.13, −0.14, −0.15,−0.16, −0.17, −0.18, −0.19, −0.2, −0.21, −0.22, −0.23, −0.24, −0.25,−0.26, −0.27, −0.28, −0.29, −0.3, −0.31, −0.32, −0.33, −0.34, −0.35,−0.36, −0.37, −0.38, −0.39, −0.4, −0.41, −0.42, −0.43, −0.44, −0.45,−0.46, −0.47, −0.48, −0.49, −0.5, −0.55, −0.6, −0.65, −0.7, −0.75, −0.8,−0.85, −0.9, −0.95, −1, −1.1, −1.2, −1.3, −1.4, −1.5, −1.6, −1.7, −1.8,−1.9, −2, −2.1, −2.2, −2.3, −2.4, −2.5, −2.6, −2.7, −2.8, −2.9, −3,−3.1, −3.2, −3.3, −3.4, −3.5, −3.6, −3.7, −3.8, −3.9, −4, −4.1, −4.2,−4.3, −4.4, −4.5, −4.6, −4.7, −4.8, −4.9, −5, −5.5, −6, −6.5, −7, −7.5,−8, −8.5, −9, −9.5, −10, −11, −12, −13, −14, −15, −16, −17, −18, −19,−20 or less, as determined by one algorithm, and wherein any segmentwhose median log 2 ratio is less than or equal to −0.1, −0.11, −0.12,−0.13, −0.14, −0.15, −0.16, −0.17, −0.18, −0.19, −0.2, −0.21, −0.22,−0.23, −0.24, −0.25, −0.26, −0.27, −0.28, −0.29, −0.3, −0.31, −0.32,−0.33, −0.34, −0.35, −0.36, −0.37, −0.38, −0.39, −0.4, −0.41, −0.42,−0.43, −0.44, −0.45, −0.46, −0.47, −0.48, −0.49, −0.5, −0.55, −0.6,−0.65, −0.7, −0.75, −0.8, −0.85, −0.9, −0.95, −1, −1.1, −1.2, −1.3,−1.4, −1.5, −1.6, −1.7, −1.8, −1.9, −2, −2.1, −2.2, −2.3, −2.4, −2.5,−2.6, −2.7, −2.8, −2.9, −3, −3.1, −3.2, −3.3, −3.4, −3.5, −3.6, −3.7,−3.8, −3.9, −4, −4.1, −4.2, −4.3, −4.4, −4.5, −4.6, −4.7, −4.8, −4.9,−5, −5.5, −6, −6.5, −7, −7.5, −8, −8.5, −9, −9.5, −10, −11, −12, −13,−14, −15, −16, −17, −18, −19, −20, or less, as determined by the otheralgorithm can be classified as a loss. For example, CNV calling cancomprise using the Aberration Detection Module 2 (ADM2) algorithm andthe DNAcopy algorithm, wherein losses can be determined according to atwo threshold log 2 ratios, wherein the Aberration Detection Module 2(ADM2) algorithm log 2 ratio can be −0.25 and the DNAcopy algorithm log2 ratio can be −0.41.

In some embodiments, the use of two algorithms to call or identifysignificant genetic variations can be a stringent method. In someembodiments, the use of two algorithms to call or identify significantgenetic variations can be a more stringent method compared to the use ofone algorithm to call or identify significant genetic variations.

In some embodiments, any CNV segment whose median log 2 ratio is greaterthan a log 2 ratio threshold value can be classified as a gain. Forexample, any segment whose median log 2 ratio is greater than 0.1, 0.11,0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23,0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35,0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47,0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, or more can be classified as a gain.

In some embodiments, one algorithm can be used to call or identifysignificant genetic variations, wherein any segment whose median log 2ratio is greater than or equal to 0.1, 0.11, 0.12, 0.13, 0.14, 0.15,0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27,0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39,0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.55,0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,or more can be classified as a gain. For example, any CNV segment whosemedian log 2 ratio is greater than 0.35 as determined by DNAcopy can beclassified as a gain. For example, gains can be determined according toa threshold log 2 ratio, which can be set at 0.35.

In some embodiments, two algorithms can be used to call or identifysignificant genetic variations, wherein any segment whose median log 2ratio is greater than or equal to 0.1, 0.11, 0.12, 0.13, 0.14, 0.15,0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27,0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39,0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5, 0.55,0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 ormore, as determined by one algorithm, and wherein any segment whosemedian log 2 ratio is greater than or equal to 0.1, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25,0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37,0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49,or 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, or more, as determined by the other algorithm the can beclassified as a gain. For example, CNV calling can comprise using theAberration Detection Module 2 (ADM2) algorithm and the DNAcopyalgorithm, wherein gains can be determined according to a two thresholdlog 2 ratios, wherein the Aberration Detection Module 2 (ADM2) algorithmlog 2 ratio can be 0.25 and the DNAcopy algorithm log 2 ratio can be0.32.

Any CNV segment whose absolute (median log-ratio/mad) value is less than2 can be excluded (not identified as a significant genetic variation).For example, any CNV segment whose absolute (median log-ratio/mad) valueis less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9,0.8, 0.7, 0.6, or 0.5 or less can be excluded.

In some embodiments, multivariate analyses or joint risk analyses,including the use of multiplicative model for overall risk assessment,can subsequently be used to determine the overall risk conferred basedon the genotype status at the multiple loci. Use of a multiplicativemodel, for example, assuming that the risk of individual risk variantsmultiply to establish the overall effect, allows for a straight-forwardcalculation of the overall risk for multiple markers. The multiplicativemodel is a parsimonious model that usually fits the data of complextraits reasonably well. Deviations from multiplicity have been rarelydescribed in the context of common variants for common diseases, and ifreported are usually only suggestive since very large sample sizes canbe required to be able to demonstrate statistical interactions betweenloci. Assessment of risk based on such analysis can subsequently be usedin the methods, uses and kits of the disclosure, as described herein.

In some embodiments, the significance of increased or decreasedsusceptibility can be measured by a percentage. In some embodiments, asignificant increased susceptibility can be measured as a relativesusceptibility of at least 1.2, including but not limited to: at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least9.0, at least 10.0, and at least 15.0. In some embodiments, a relativesusceptibility of at least 2.0, at least 3.0, at least 4.0, at least,5.0, at least 6.0, or at least 10.0 is significant. Other values forsignificant susceptibility are also contemplated, for example, at least2.5, 3.5, 4.5, 5.5, or any suitable other numerical values, wherein thevalues are also within scope of the present disclosure. In someembodiments, a significant increase in susceptibility is at least about20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000%, and 1500%. In one particularembodiment, a significant increase in susceptibility is at least 100%.In other embodiments, a significant increase in susceptibility is atleast 200%, at least 300%, at least 400%, at least 500%, at least 700%,at least 800%, at least 900% and at least 1000%. Other cutoffs or rangesas deemed suitable by the person skilled in the art to characterize thedisclosure are also contemplated, and those are also within scope of thepresent disclosure. In certain embodiments, a significant increase insusceptibility is characterized by a p-value, such as a p-value of lessthan 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1,less than 0.05, less than 0.01, less than 0.001, less than 0.0001, lessthan 0.00001, less than 0.000001, less than 0.0000001, less than0.00000001, or less than 0.000000001.

In some embodiments, an individual who is at a decreased susceptibilityfor or the lack of presence of a developmental condition can be anindividual in whom at least one genetic variation, conferring decreasedsusceptibility for or the lack of presence of the developmental disorderis identified. In some embodiments, the genetic variations conferringdecreased susceptibility are also protective. In one aspect, the geneticvariations can confer a significant decreased susceptibility of or lackof presence of the developmental disorder.

In some embodiments, significant decreased susceptibility can bemeasured as a relative susceptibility of less than 0.9, including butnot limited to less than 0.9, less than 0.8, less than 0.7, less than0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and lessthan 0.1. In some embodiments, the decrease in susceptibility is atleast 20%, including but not limited to at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% and at least 98%. Other cutoffs orranges as deemed suitable by the person, skilled in the art tocharacterize the disclosure are however also contemplated, and those arealso within scope of the present disclosure. In certain embodiments, asignificant decrease in susceptibility is characterized by a p-value,such as a p-value of less than 0.05, less than 0.01, less than 0.001,less than 0.0001, less than 0.00001, less than 0.000001, less than0.0000001, less than 0.00000001, or less than 0.000000001. Other testsfor significance can be used, for example, a Fisher-exact test. Otherstatistical tests of significance known to the skilled person are alsocontemplated and are also within scope of the disclosure.

In some preferred embodiments, the significance of increased ordecreased susceptibility can be determined according to the ratio ofmeasurements from a test subject to a reference subject. In someembodiments, losses or gains of one or more CNVs can be determinedaccording to a threshold log₂ ratio determined by these measurements. Insome embodiments, a log₂ ratio value greater than 0.35 is indicative ofa gain of one or more CNVs. In some embodiments, a log₂ ratio value lessthan −0.35 is indicative of a loss of one or more CNVs. In someembodiments, the ratio of measurements from a test subject to areference subject may be inverted such that the log 2 ratios of copynumber gains are negative and the log 2 ratios of copy number losses arepositive.

In some embodiments, the combined or overall susceptibility associatedwith a plurality of variants associated with a developmental disordercan also be assessed; for example, the genetic variations describedherein to be associated with susceptibility to a developmental disordercan be combined with other common genetic risk factors. Combined riskfor such genetic variants can be estimated in an analogous fashion tothe methods described herein.

Calculating risk conferred by a particular genotype for the individualcan be based on comparing the genotype of the individual to previouslydetermined risk expressed, for example, as a relative risk (RR) or anodds ratio (OR), for the genotype, for example, for a heterozygouscarrier of an at-risk variant for a developmental disorder. An oddsratio can be a statistical measure used as a metric of causality. Forexample, in genetic disease research it can be used to convey thesignificance of a variant in a disease cohort relative to anunaffected/normal cohort. The calculated risk for the individual can bethe relative risk for a subject, or for a specific genotype of asubject, compared to the average population. The average population riskcan be expressed as a weighted average of the risks of differentgenotypes, using results from a reference population, and theappropriate calculations to calculate the risk of a genotype grouprelative to the population can then be performed. Alternatively, therisk for an individual can be based on a comparison of particulargenotypes, for example, heterozygous and/or homozygous carriers of anat-risk allele of a marker compared with non-carriers of the at-riskallele. Using the population average can, in certain embodiments, bemore convenient, since it provides a measure that can be easy tointerpret for the user, for example, a measure that gives the risk forthe individual, based on his/her genotype, compared with the average inthe population.

In some embodiments, the OR value can be calculated as follows:OR=(A/(N1−A))/(U/(N2−U)), where A=number of affected cases with variant,N1=total number of affected cases, U=number of unaffected cases withvariant and N2=total number of unaffected cases. In circumstances whereU=0, it is conventional to set U=1, so as to avoid infinities In somepreferred embodiments the OR can be calculated essentially as above,except that where U OR A=0, 0.5 is added to all of A, N1, U, N2. Inanother embodiment, a Fisher's Exact Test (FET) can be calculated usingstandard methods. In another embodiment, the p-values can be correctedfor false discovery rate (FDR) using the Benjamini-Hochberg method(Benjamini Y. and Hochberg Y. 1995 J. Royal Statistical Society 57:289;Osborne J. A. and Barker C. A. 2007).

In certain embodiments of the disclosure, a genetic variation iscorrelated to a developmental disorder by referencing genetic variationdata to a look-up table that comprises correlations between the geneticvariation and a developmental disorder. The genetic variation in certainembodiments comprises at least one indication of the genetic variation.In some embodiments, the table comprises a correlation for one geneticvariation. In other embodiments, the table comprises a correlation for aplurality of genetic variations In both scenarios, by referencing to alook-up table that gives an indication of a correlation between agenetic variation and a developmental disorder, a risk for adevelopmental disorder, or a susceptibility to a developmental disorder,can be identified in the individual from whom the nucleic acid sample isderived.

The present disclosure also pertains to methods of clinical screening,for example, diagnosis, prognosis, or theranosis of a subject performedby a medical professional using the methods disclosed herein. In otherembodiments, the disclosure pertains to methods of screening performedby a layman. The layman can be a customer of a genotyping, microarray,exome sequencing, or whole genome sequencing service provider. Thelayman can also be a genotype, microarray, exome sequencing, or wholegenome sequencing service provider, who performs genetic analysis on aDNA sample from an individual, in order to provide service related togenetic risk factors for particular traits or diseases, based on thegenotype status of the subject obtained from use of the methodsdescribed herein. The resulting genotype or genetic information can bemade available to the individual and can be compared to informationabout developmental disorders or risk of developing a developmentaldisorder associated with one or various genetic variations, includingbut not limited to, information from public or private genetic variationdatabases or literature and scientific publications. The screeningapplications of developmental disorder-associated genetic variations, asdescribed herein, can, for example, be performed by an individual, ahealth professional, or a third party, for example a service providerwho interprets genotype information from the subject. In someembodiments the genetic analysis is performed in a CLIA-certifiedlaboratory (i.e., the federal regulatory standards the U.S. that arespecified in the Clinical Laboratory Improvement Amendments,administered by the Centers for Medicare and Medicaid Services) orequivalent laboratories in Europe and elsewhere in the world.

The information derived from analyzing sequence data can be communicatedto any particular body, including the individual from which the nucleicacid sample or sequence data is derived, a guardian or representative ofthe individual, clinician, research professional, medical professional,service provider, and medical insurer or insurance company. Medicalprofessionals can be, for example, doctors, nurses, medical laboratorytechnologists, and pharmacists. Research professionals can be, forexample, principle investigators, research technicians, postdoctoraltrainees, and graduate students.

In some embodiments, a professional can be assisted by determiningwhether specific genetic variants are present in a nucleic acid samplefrom a subject, and communicating information about genetic variants toa professional. After information about specific genetic variants isreported, a medical professional can take one or more actions that canaffect subject care. For example, a medical professional can recordinformation in the subject's medical record regarding the subject's riskof developing a developmental disorder. In some embodiments, a medicalprofessional can record information regarding risk assessment, orotherwise transform the subject's medical record, to reflect thesubject's current medical condition. In some embodiments, a medicalprofessional can review and evaluate a subject's entire medical recordand assess multiple treatment strategies for clinical intervention of asubject's condition.

A medical professional can initiate or modify treatment after receivinginformation regarding a subject's screening of a developmental disorder,for example. In some embodiments, a medical professional can recommend achange in therapy. In some embodiments, a medical professional canenroll a subject in a clinical trial for, by way of example, detectingcorrelations between a haplotype as described herein and any measurableor quantifiable parameter relating to the outcome of the treatment asdescribed above.

In some embodiments, a medical professional can communicate informationregarding a subject's screening of developing a developmental disorderto a subject or a subject's family. In some embodiments, a medicalprofessional can provide a subject and/or a subject's family withinformation regarding a developmental disorder and risk assessmentinformation, including treatment options, and referrals to specialists.In some embodiments, a medical professional can provide a copy of asubject's medical records to a specialist. In some embodiments, aresearch professional can apply information regarding a subject's riskof developing a developmental disorder to advance scientific research.In some embodiments, a research professional can obtain a subject'shaplotype as described herein to evaluate a subject's enrollment, orcontinued participation, in a research study or clinical trial. In someembodiments, a research professional can communicate informationregarding a subject's screening of a developmental disorder to a medicalprofessional. In some embodiments, a research professional can refer asubject to a medical professional.

Any appropriate method can be used to communicate information to anotherperson. For example, information can be given directly or indirectly toa professional and a laboratory technician can input a subject's geneticvariation as described herein into a computer-based record. In someembodiments, information is communicated by making a physical alterationto medical or research records. For example, a medical professional canmake a permanent notation or flag a medical record for communicating therisk assessment to other medical professionals reviewing the record. Inaddition, any type of communication can be used to communicate the riskassessment information. For example, mail, e-mail, telephone, andface-to-face interactions can be used. The information also can becommunicated to a professional by making that information electronicallyavailable to the professional. For example, the information can becommunicated to a professional by placing the information on a computerdatabase such that the professional can access the information. Inaddition, the information can be communicated to a hospital, clinic, orresearch facility serving as an agent for the professional.

Results of these tests, and optionally interpretive information, can bereturned to the subject, the health care provider or to a third party.The results can be communicated to the tested subject, for example, witha prognosis and optionally interpretive materials that can help thesubject understand the test results and prognosis; used by a health careprovider, for example, to determine whether to administer a specificdrug, or whether a subject should be assigned to a specific category,for example, a category associated with a specific diseaseendophenotype, or with drug response or non-response; used by a thirdparty such as a healthcare payer, for example, an insurance company orHMO, or other agency, to determine whether or not to reimburse a healthcare provider for services to the subject, or whether to approve theprovision of services to the subject. For example, the healthcare payercan decide to reimburse a health care provider for treatments for adevelopmental disorder if the subject has a developmental disorder orhas an increased risk of developing a developmental disorder.

Also provided herein are databases that include a list of geneticvariations as described herein, and wherein the list can be largely orentirely limited to genetic variations identified as useful forscreening a developmental disorder as described herein. The list can bestored, for example, on a flat file or computer-readable medium. Thedatabases can further include information regarding one or moresubjects, for example, whether a subject is affected or unaffected,clinical information such as endophenotype, age of onset of symptoms,any treatments administered and outcomes, for example, data relevant topharmacogenomics, diagnostics, prognostics or theranostics, and otherdetails, for example, data about the disorder in the subject, orenvironmental or other genetic factors. The databases can be used todetect correlations between a particular haplotype and the informationregarding the subject.

The methods described herein can also include the generation of reportsfor use, for example, by a subject, care giver, or researcher, thatinclude information regarding a subject's genetic variations, andoptionally further information such as treatments administered,treatment history, medical history, predicted response, and actualresponse. The reports can be recorded in a tangible medium, e.g., acomputer-readable disk, a solid state memory device, or an opticalstorage device.

Methods of Screening Using Variations in RNA and/or Polypeptides

In some embodiments of the disclosure, screening of a developmentaldisorder can be made by examining or comparing changes in expression,localization, binding partners, and composition of a polypeptide encodedby a nucleic acid associated with a developmental disorder, for example,in those instances where the genetic variations of the presentdisclosure results in a change in the composition or expression of thepolypeptide and/or RNA, for example, mRNAs, microRNAs (miRNAs), andother noncoding RNAs (ncRNAs). Thus, screening of a developmentaldisorder can be made by examining expression and/or composition of oneof these polypeptides and/or RNA, or another polypeptide and/or RNAencoded by a nucleic acid associated with a developmental disorder, inthose instances where the genetic variation of the present disclosureresults in a change in the expression, localization, binding partners,and/or composition of the polypeptide and/or RNA. In some embodiments,screening can comprise diagnosing a subject. In some embodiments,screening can comprise determining a prognosis of a subject, for exampledetermining the susceptibility of developing a developmental disorder.In some embodiments, screening can comprise theranosing a subject.

The genetic variations described herein that show association to adevelopmental disorder can play a role through their effect on one ormore of these nearby genes. For example, while not intending to belimited by theory, it is generally expected that a deletion of achromosomal segment comprising a particular gene, or a fragment of agene, can either result in an altered composition or expression, orboth, of the encoded polypeptide and/or mRNA. Likewise, duplications, orhigh number copy number variations, are in general expected to result inincreased expression of encoded polypeptide and/or RNA. Other possiblemechanisms affecting genes within a genetic variation region include,for example, effects on transcription, effects on RNA splicing,alterations in relative amounts of alternative splice forms of mRNA,effects on RNA stability, effects on transport from the nucleus tocytoplasm, and effects on the efficiency and accuracy of translation.Thus, DNA variations can be detected directly, using the subjectsunamplified or amplified genomic DNA, or indirectly, using RNA or DNAobtained from the subject's tissue(s) that are present in an aberrantform or expression level as a result of the genetic variations of thedisclosure showing association to a developmental disorder (e.g., ASD).In another embodiment, DNA variations can be detected indirectly using apolypeptide or protein obtained from the subject's tissue(s) that ispresent in an aberrant form or expression level as a result of geneticvariations of the disclosure showing association to the developmentaldisorder. In another embodiment, an aberrant form or expression level ofa polypeptide or protein that results from one or more geneticvariations of the disclosure showing association to the developmentaldisorder can be detected indirectly via another polypeptide or proteinpresent in the same biological/cellular pathway that is modulated orinteracts with said polypeptide or protein that results from one or moregenetic variations of the disclosure. In some embodiments, the geneticvariations of the disclosure showing association to a developmentaldisorder can affect the expression of a gene within the geneticvariation region. In some embodiments, a genetic variation affecting anexonic region of a gene can affect, disrupt, or modulate the expressionof the gene. In some embodiments, a genetic variation affecting anintergenic region of a gene can affect, disrupt, or modulate theexpression of the gene.

Certain genetic variation regions can have flanking duplicated segments,and genes within such segments can have altered expression and/orcomposition as a result of such genomic alterations. Regulatory elementsaffecting gene expression can be located far away, even as far as tensor hundreds of kilobases away, from the gene that is regulated by saidregulatory elements. Thus, in some embodiments, regulatory elements forgenes that are located outside the genetic variation region can belocated within the genetic variation, and thus be affected by thegenetic variation. It is thus contemplated that the detection of thegenetic variations described herein, can be used for assessingexpression for one or more of associated genes not directly impacted bythe genetic variations. In some embodiments, a genetic variationaffecting an intergenic region of a gene can affect, disrupt, ormodulate the expression of a gene located elsewhere in the genome, suchas described above. For example, a genetic variation affecting anintergenic region of a gene can affect, disrupt, or modulate theexpression of a transcription factor, located elsewhere in the genome,which regulates the gene.

In some embodiments, genetic variations of the disclosure showingassociation to ASD can affect protein expression at the translationallevel. It can be appreciated by those skilled in the art that this canoccur by increased or decreased expression of one or more microRNAs(miRNAs) that regulates expression of a protein known to be important,or implicated, in the cause, onset, or progression of ASD. Increased ordecreased expression of the one or more miRNAs can result from gain orloss of the whole miRNA gene, disruption or impairment of a portion ofthe gene (e.g., by an indel or CNV), or even a single base change (SNPor SNV) that produces an altered, non-functional or aberrant functioningmiRNA sequence. It can also be appreciated by those skilled in the artthat the expression of protein, for example, one known to cause ASD byincreased or decreased expression, can result due to a genetic variationthat results in alteration of an existing miRNA binding site within thepolypeptide's mRNA transcript, or even creates a new miRNA binding sitethat leads to aberrant polypeptide expression.

A variety of methods can be used for detecting polypeptide compositionand/or expression levels, including but not limited to enzyme linkedimmunosorbent assays (ELISA), Western blots, spectroscopy, massspectrometry, peptide arrays, colorimetry, electrophoresis, isoelectricfocusing, immunoprecipitations, immunoassays, and immunofluorescence andother methods well-known in the art. A test nucleic acid sample from asubject can be assessed for the presence of an alteration in theexpression and/or an alteration in composition of the polypeptideencoded by a nucleic acid associated with a developmental disorder. An“alteration” in the polypeptide expression or composition, as usedherein, refers to an alteration in expression or composition in a testnucleic acid sample, as compared to the expression or composition of thepolypeptide in a control nucleic acid sample. Such alteration can, forexample, be an alteration in the quantitative polypeptide expression orcan be an alteration in the qualitative polypeptide expression, forexample, expression of a mutant polypeptide or of a different splicingvariant, or a combination thereof. In some embodiments, screening of adevelopmental disorder can be made by detecting a particular splicingvariant encoded by a nucleic acid associated with a developmentaldisorder, or a particular pattern of splicing variants.

Antibodies can be polyclonal or monoclonal and can be labeled orunlabeled. An intact antibody or a fragment thereof can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling adetectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled as previously described herein. Othernon-limiting examples of indirect labeling include detection of aprimary antibody using a labeled secondary antibody, for example, afluorescently-labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently-labeledstreptavidin.

Detecting Genetic Variations Associated with Autism Spectrum Disorder

Described herein, are methods that can be used to detect geneticvariations. Detecting specific genetic variations, for examplepolymorphic markers and/or haplotypes, copy number, absence or presenceof an allele, or genotype associated with a developmental disorder asdescribed herein, can be accomplished by methods known in the art foranalyzing nucleic acids and/or detecting sequences at polymorphic orgenetically variable sites, for example, amplification techniques,hybridization techniques, sequencing, arrays, or any combinationthereof. Thus, by use of these methods disclosed herein or other methodsavailable to the person skilled in the art, one or more alleles atpolymorphic markers, including microsatellites, SNPs, SNVs, indels,CNVs, or other types of genetic variations, can be identified in asample obtained from a subject.

Nucleic Acids

The nucleic acids and polypeptides described herein can be used inmethods and kits of the present disclosure. In some embodiments,aptamers that specifically bind the nucleic acids and polypeptidesdescribed herein can be used in methods and kits of the presentdisclosure. As used herein, a nucleic acid can comprise adeoxyribonucleotide (DNA) or ribonucleotide (RNA), whether singular orin polymers, naturally occurring or non-naturally occurring,double-stranded or single-stranded, coding, for example a translatedgene, or non-coding, for example a regulatory region, or any fragments,derivatives, mimetics or complements thereof. In some embodiments,nucleic acids can comprise oligonucleotides, nucleotides,polynucleotides, nucleic acid sequences, genomic sequences,complementary DNA (cDNA), antisense nucleic acids, DNA regions, probes,primers, genes, regulatory regions, introns, exons, open-reading frames,binding sites, target nucleic acids and allele-specific nucleic acids.

A “probe,” as used herein, includes a nucleic acid fragment forexamining a nucleic acid in a specimen using the hybridization reactionbased on the complementarity of nucleic acid.

“A “hybrid” as used herein, includes a double strand formed between anyone of the abovementioned nucleic acid, within the same type, or acrossdifferent types, including DNA-DNA, DNA-RNA, RNA-RNA or the like.

“Isolated” nucleic acids, as used herein, are separated from nucleicacids that normally flank the gene or nucleotide sequence (as in genomicsequences) and/or has been completely or partially purified from othertranscribed sequences (e.g., as in an RNA library). For example,isolated nucleic acids of the disclosure can be substantially isolatedwith respect to the complex cellular milieu in which it naturallyoccurs, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized. Insome instances, the isolated material can form part of a composition,for example, a crude extract containing other substances, buffer systemor reagent mix. In some embodiments, the material can be purified toessential homogeneity using methods known in the art, for example, bypolyacrylamide gel electrophoresis (PAGE) or column chromatography(e.g., HPLC). With regard to genomic DNA (gDNA), the term “isolated”also can refer to nucleic acids that are separated from the chromosomewith which the genomic DNA is naturally associated. For example, theisolated nucleic acid molecule can contain less than about 250 kb, 200kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb,1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acidmolecule in the gDNA of the cell from which the nucleic acid molecule isderived.

Nucleic acids can be fused to other coding or regulatory sequences canbe considered isolated. For example, recombinant DNA contained in avector is included in the definition of “isolated” as used herein. Insome embodiments, isolated nucleic acids can include recombinant DNAmolecules in heterologous host cells or heterologous organisms, as wellas partially or substantially purified DNA molecules in solution.Isolated nucleic acids also encompass in vivo and in vitro RNAtranscripts of the DNA molecules of the present disclosure. An isolatednucleic acid molecule or nucleotide sequence can be synthesizedchemically or by recombinant means. Such isolated nucleotide sequencescan be useful, for example, in the manufacture of the encodedpolypeptide, as probes for isolating homologous sequences (e.g., fromother mammalian species), for gene mapping (e.g., by in situhybridization with chromosomes), or for detecting expression of thegene, in tissue (e.g., human tissue), such as by Northern blot analysisor other hybridization techniques disclosed herein. The disclosure alsopertains to nucleic acid sequences that hybridize under high stringencyhybridization conditions, such as for selective hybridization, to anucleotide sequence described herein Such nucleic acid sequences can bedetected and/or isolated by allele- or sequence-specific hybridization(e.g., under high stringency conditions). Stringency conditions andmethods for nucleic acid hybridizations are well known to the skilledperson (see, e.g., Current Protocols in Molecular Biology, Ausubel, F.et al., John Wiley & Sons, (1998), and Kraus, M. and Aaronson, S.,Methods Enzymol., 200:546-556 (1991), the entire teachings of which areincorporated by reference herein.

Calculations of “identity” or “percent identity” between two or morenucleotide or amino acid sequences can be determined by aligning thesequences for optimal comparison purposes (e.g., gaps can be introducedin the sequence of a first sequence). The nucleotides at correspondingpositions are then compared, and the percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=#of identical positions/total #ofpositions x 100). For example, a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

In some embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95%, of the length ofthe reference sequence. The actual comparison of the two sequences canbe accomplished by well-known methods, for example, using a mathematicalalgorithm. A non-limiting example of such a mathematical algorithm isdescribed in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA,90-5873-5877 (1993). Such an algorithm is incorporated into the NBLASTand XBLAST programs (version 2.0), as described in Altschul, S. et al.,Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and GappedBLAST programs, any relevant parameters of the respective programs(e.g., NBLAST) can be used. For example, parameters for sequencecomparison can be set at score=100, word length=12, or can be varied(e.g., W=5 or W=20). Other examples include the algorithm of Myers andMiller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In someembodiments, the percent identity between two amino acid sequences canbe accomplished using, for example, the GAP program in the GCG softwarepackage (Accelrys, Cambridge, UK).

“Probes” or “primers” can be oligonucleotides that hybridize in abase-specific manner to a complementary strand of a nucleic acidmolecule. Probes can include primers, which can be a single-strandedoligonucleotide probe that can act as a point of initiation oftemplate-directed DNA synthesis using methods including but not limitedto, polymerase chain reaction (PCR) and ligase chain reaction (LCR) foramplification of a target sequence. Oligonucleotides, as describedherein, can include segments or fragments of nucleic acid sequences, ortheir complements. In some embodiments, DNA segments can be between 5and 10,000 contiguous bases, and can range from 5, 10, 12, 15, 20, or 25nucleotides to 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, 1000 or 10,000nucleotides. In addition to DNA and RNA, probes and primers can includepolypeptide nucleic acids (PNA), as described in Nielsen, P. et al.,Science 254: 1497-1500 (1991). A probe or primer can comprise a regionof nucleotide sequence that hybridizes to at least about 15, typicallyabout 20-25, and in certain embodiments about 40, 50, 60 or 75,consecutive nucleotides of a nucleic acid molecule.

The present disclosure also provides isolated nucleic acids, forexample, probes or primers, that contain a fragment or portion that canselectively hybridize to a nucleic acid that comprises, or consists of,a nucleotide sequence, wherein the nucleotide sequence can comprise atleast one polymorphism or polymorphic allele contained in the geneticvariations described herein or the wild-type nucleotide that is locatedat the same position, or the compliments thereof. In some embodiments,the probe or primer can be at least 70% identical, at least 80%identical, at least 85% identical, at least 90% identical, or at least95% identical, to the contiguous nucleotide sequence or to thecomplement of the contiguous nucleotide sequence.

In some embodiments, a nucleic acid probe can be an oligonucleotidecapable of hybridizing with a complementary region of a gene associatedwith a developmental disorder containing a genetic variation describedherein. The nucleic acid fragments of the disclosure can be used asprobes or primers in assays such as those described herein.

The nucleic acids of the disclosure, such as those described above, canbe identified and isolated using standard molecular biology techniqueswell known to the skilled person. In some embodiments, DNA can beamplified and/or can be labeled (e.g., radiolabeled, fluorescentlylabeled) and used as a probe for screening, for example, a cDNA libraryderived from an organism. cDNA can be derived from mRNA and can becontained in a suitable vector. For example, corresponding clones can beisolated, DNA obtained fallowing in vivo excision, and the cloned insertcan be sequenced in either or both orientations by art-recognizedmethods to identify the correct reading frame encoding a polypeptide ofthe appropriate molecular weight. Using these or similar methods, thepolypeptide and the DNA encoding the polypeptide can be isolated,sequenced and further characterized.

In some embodiments, nucleic acid can comprise one or morepolymorphisms, variations, or mutations, for example, single nucleotidepolymorphisms (SNPs), single nucleotide variations (SNVs), copy numbervariations (CNVs), for example, insertions, deletions, inversions, andtranslocations. In some embodiments, nucleic acids can comprise analogs,for example, phosphorothioates, phosphoramidates, methyl phosphonate,chiralmethyl phosphonates, 2-O-methyl ribonucleotides, or modifiednucleic acids, for example, modified backbone residues or linkages, ornucleic acids combined with carbohydrates, lipids, polypeptide or othermaterials, or peptide nucleic acids (PNAs), for example, chromatin,ribosomes, and transcriptosomes. In some embodiments nucleic acids cancomprise nucleic acids in various structures, for example, A DNA, B DNA,Z-form DNA, siRNA, tRNA, and ribozymes. In some embodiments, the nucleicacid may be naturally or non-naturally polymorphic, for example, havingone or more sequence differences, for example, additions, deletionsand/or substitutions, as compared to a reference sequence. In someembodiments, a reference sequence can be based on publicly availableinformation, for example, the U.C. Santa Cruz Human Genome BrowserGateway (genome.ucsc.edu/cgi-bin/hgGateway) or the NCBI website(www.ncbi.nlm.nih.gov). In some embodiments, a reference sequence can bedetermined by a practitioner of the present disclosure using methodswell known in the art, for example, by sequencing a reference nucleicacid.

In some embodiment a probe can hybridize to an allele, SNP, or CNV asdescribed herein. In some embodiments, the probe can bind to anothermarker sequence associated with a developmental disorder as describedherein.

One of skill in the art would know how to design a probe so thatsequence specific hybridization can occur only if a particular allele ispresent in a genomic sequence from a test nucleic acid sample. Thedisclosure can also be reduced to practice using any convenientgenotyping method, including commercially available technologies andmethods for genotyping particular genetic variations

Control probes can also be used, for example, a probe that binds a lessvariable sequence, for example, a repetitive DNA associated with acentromere of a chromosome, can be used as a control. In someembodiments, probes can be obtained from commercial sources. In someembodiments, probes can be synthesized, for example, chemically or invitro, or made from chromosomal or genomic DNA through standardtechniques. In some embodiments sources of DNA that can be used includegenomic DNA, cloned DNA sequences, somatic cell hybrids that containone, or a part of one, human chromosome along with the normal chromosomecomplement of the host, and chromosomes purified by flow cytometry ormicrodissection. The region of interest can be isolated through cloning,or by site-specific amplification using PCR.

One or more nucleic acids for example, a probe or primer, can also belabeled, for example, by direct labeling, to comprise a detectablelabel. A detectable label can comprise any label capable of detection bya physical, chemical, or a biological process for example, a radioactivelabel, such as ³²P or ³H, a fluorescent label, such as FITC, achromophore label, an affinity-ligand label, an enzyme label, such asalkaline phosphatase, horseradish peroxidase, or 12 galactosidase, anenzyme cofactor label, a hapten conjugate label, such as digoxigenin ordinitrophenyl, a Raman signal generating label, a magnetic label, a spinlabel, an epitope label, such as the FLAG or HA epitope, a luminescentlabel, a heavy atom label, a nanoparticle label, an electrochemicallabel, a light scattering label, a spherical shell label, semiconductornanocrystal label, such as quantum dots (described in U.S. Pat. No.6,207,392), and probes labeled with any other signal generating labelknown to those of skill in the art, wherein a label can allow the probeto be visualized with or without a secondary detection molecule. Anucleotide can be directly incorporated into a probe with standardtechniques, for example, nick translation, random priming, and PCRlabeling. A “signal,” as used herein, include a signal suitablydetectable and measurable by appropriate means, including fluorescence,radioactivity, chemiluminescence, and the like.

Non-limiting examples of label moieties useful for detection include,without limitation, suitable enzymes such as horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;members of a binding pair that are capable of forming complexes such asstreptavidin/biotin, avidin/biotin or an antigen/antibody complexincluding, for example, rabbit IgG and anti-rabbit IgG; fluorophoressuch as umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein,erythrosin, coumarin, methyl coumarin, pyrene, malachite green,stilbene, lucifer yellow, Cascade Blue, Tex. Red, dichlorotriazinylaminefluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanidecomplexes such as those including Europium and Terbium, cyanine dyefamily members, such as Cy3 and Cy5, molecular beacons and fluorescentderivatives thereof, as well as others known in the art as described,for example, in Principles of Fluorescence Spectroscopy, Joseph R.Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6thEdition of the Molecular Probes Handbook by Richard P. Hoagland; aluminescent material such as luminol; light scattering or plasmonresonant materials such as gold or silver particles or quantum dots; orradioactive material include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, Tc99m, ³²P, ³³P, ³⁵Sor ³H.

Other labels can also be used in the methods of the present disclosure,for example, backbone labels. Backbone labels comprise nucleic acidstains that bind nucleic acids in a sequence independent manner.Non-limiting examples include intercalating dyes such as phenanthridinesand acridines (e.g., ethidium bromide, propidium iodide, hexidiumiodide, dihydroethidium, ethidium homodimer-1 and -2, ethidiummonoazide, and ACMA); some minor grove binders such as indoles andimidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI);and miscellaneous nucleic acid stains such as acridine orange (alsocapable of intercalating), 7-AAD, actinomycin D, LDS751, andhydroxystilbamidine. All of the aforementioned nucleic acid stains arecommercially available from suppliers such as Molecular Probes, Inc.Still other examples of nucleic acid stains include the following dyesfrom Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green,SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1,LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3,TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3,PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II,SYBR DX, SYTO-40, −41, −42, −43, −44, −45 (blue), SYTO-13, −16, −24,−21, −23, −12, −11, −20, −22, −15, −14, −25 (green), SYTO-81, −80, −82,−83, −84, −85 (orange), SYTO-64, −17, −59, −61, −62, −60, −63 (red).

In some embodiments, fluorophores of different colors can be chosen, forexample, 7-amino-4-methylcoumarin-3-acetic acid (AMCA),5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B,5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, TRITC,rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7,Texas Red, Phar-Red, allophycocyanin (APC), and CASCADE™ blueacetylazide, such that each probe in or not in a set can be distinctlyvisualized. In some embodiments, fluorescently labeled probes can beviewed with a fluorescence microscope and an appropriate filter for eachfluorophore, or by using dual or triple band-pass filter sets to observemultiple fluorophores. In some embodiments, techniques such as flowcytometry can be used to examine the hybridization pattern of theprobes.

In other embodiments, the probes can be indirectly labeled, for example,with biotin or digoxygenin, or labeled with radioactive isotopes such as³²P and/or ³H. As a non-limiting example, a probe indirectly labeledwith biotin can be detected by avidin conjugated to a detectable marker.For example, avidin can be conjugated to an enzymatic marker such asalkaline phosphatase or horseradish peroxidase. In some embodiments,enzymatic markers can be detected using colorimetric reactions using asubstrate and/or a catalyst for the enzyme. In some embodiments,catalysts for alkaline phosphatase can be used, for example,5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. In someembodiments, a catalyst can be used for horseradish peroxidase, forexample, diaminobenzoate.

Methods of Detecting Genetic Variations

In some embodiments, standard techniques for genotyping for the presencegenetic variations, for example, amplification, can be used.Amplification of nucleic acids can be accomplished using methods knownin the art. Generally, sequence information from the region of interestcan be used to design oligonucleotide primers that can be identical orsimilar in sequence to opposite strands of a template to be amplified.In some embodiments, amplification methods can include but are notlimited to, fluorescence-based techniques utilizing PCR, for example,ligase chain reaction (LCR), Nested PCR, transcription amplification,self-sustained sequence replication, nucleic acid based sequenceamplification (NASBA), and multiplex ligation-dependent probeamplification (MLPA). Guidelines for selecting primers for PCRamplification are well known in the art. In some embodiments, a computerprogram can be used to design primers, for example, Oligo (NationalBiosciences, Inc, Plymouth Minn.), MacVector (Kodak/IBI), and GCG suiteof sequence analysis programs.

In some embodiments, commercial methodologies available for genotyping,for example, SNP genotyping, can be used, but are not limited to, TaqMangenotyping assays (Applied Biosystems), SNPlex platforms (AppliedBiosystems), gel electrophoresis, capillary electrophoresis, sizeexclusion chromatography, mass spectrometry, for example, MassARRAYsystem (Sequenom), minisequencing methods, real-time Polymerase ChainReaction (PCR), Bio-Plex system (BioRad), CEQ and SNPstream systems(Beckman), array hybridization technology, for example, AffymetrixGeneChip (Perlegen), BeadArray Technologies, for example, IlluminaGoldenGate and Infinium assays, array tag technology, MultiplexLigation-dependent Probe Amplification (MLPA), and endonuclease-basedfluorescence hybridization technology (Invader; Third Wave). PCR can bea procedure in which target nucleic acid is amplified in a mannersimilar to that described in U.S. Pat. No. 4,683,195 and subsequentmodifications of the procedure described therein. PCR can include athree phase temperature cycle of denaturation of DNA into singlestrands, annealing of primers to the denatured strands, and extension ofthe primers by a thermostable DNA polymerase enzyme. This cycle can berepeated so that there are enough copies to be detected and analyzed. Insome embodiments, real-time quantitative PCR can be used to determinegenetic variations, wherein quantitative PCR can permit both detectionand quantification of a DNA sequence in a nucleic acid sample, forexample, as an absolute number of copies or as a relative amount whennormalized to DNA input or other normalizing genes. In some embodiments,methods of quantification can include the use of fluorescent dyes thatcan intercalate with double-stranded DNA, and modified DNAoligonucleotide probes that can fluoresce when hybridized with acomplementary DNA.

In some embodiments of the disclosure, a nucleic acid sample obtainedfrom the subject can be collected and PCR can used to amplify a fragmentof nucleic acid that comprises one or more genetic variations that canbe indicative of a susceptibility to a developmental disorder. In someembodiments, detection of genetic variations can be accomplished byexpression analysis, for example, by using quantitative PCR. In someembodiments, this technique can assess the presence or absense of agenetic alteration in the expression or composition of one or morepolypeptides or splicing variants encoded by a nucleic acid associatedwith a developmental disorder.

In some embodiments, the nucleic acid sample from a subject containing aSNP can be amplified by PCR prior to detection with a probe. In such anembodiment, the amplified DNA serves as the template for a detectionprobe and, in some embodiments, an enhancer probe. Certain embodimentsof the detection probe, the enhancer probe, and/or the primers used foramplification of the template by PCR can comprise the use of modifiedbases, for example, modified A, T, C, G, and U, wherein the use ofmodified bases can be useful for adjusting the melting temperature ofthe nucleotide probe and/or primer to the template DNA, In someembodiments, modified bases are used in the design of the detectionnucleotide probe. Any modified base known to the skilled person can beselected in these methods, and the selection of suitable bases is wellwithin the scope of the skilled person based on the teachings herein andknown bases available from commercial sources as known to the skilledperson.

In some embodiments, identification of genetic variations can beaccomplished using hybridization methods. The presence of a specificmarker allele or a particular genomic segment comprising a geneticvariation, or representative of a genetic variation, can be indicated bysequence-specific hybridization of a nucleic acid probe specific for theparticular allele or the genetic variation in a nucleic acid sample thathas or has not been amplified but methods described herein. The presenceof more than one specific marker allele or several genetic variationscan be indicated by using two or more sequence-specific nucleic acidprobes, wherein each is specific for a particular allele and/or geneticvariation.

Hybridization can be performed by methods well known to the personskilled in the art, for example, hybridization techniques such asfluorescent in situ hybridization (FISH), Southern analysis, Northernanalysis, or in situ hybridization. In some embodiments, hybridizationrefers to specific hybridization, wherein hybridization can be performedwith no mismatches. Specific hybridization, if present, can be usingstandard methods. In some embodiments, if specific hybridization occursbetween a nucleic acid probe and the nucleic acid in the nucleic acidsample, the nucleic acid sample can contain a sequence that can becomplementary to a nucleotide present in the nucleic acid probe. In someembodiments, if a nucleic acid probe can contain a particular allele ofa polymorphic marker, or particular alleles for a plurality of markers,specific hybridization is indicative of the nucleic acid beingcompletely complementary to the nucleic acid probe, including theparticular alleles at polymorphic markers within the probe. In someembodiments a probe can contain more than one marker alleles of aparticular haplotype, for example, a probe can contain allelescomplementary to 2, 3, 4, 5 or all of the markers that make up aparticular haplotype. In some embodiments detection of one or moreparticular markers of the haplotype in the nucleic acid sample isindicative that the source of the nucleic acid sample has the particularhaplotype.

In some embodiments, PCR conditions and primers can be developed thatamplify a product only when the variant allele is present or only whenthe wild type allele is present, for example, allele-specific PCR. Insome embodiments of allele-specific PCR, a method utilizing a detectionoligonucleotide probe comprising a fluorescent moiety or group at its 3′terminus and a quencher at its 5′ terminus, and an enhanceroligonucleotide, can be employed, as described by Kutyavin et al.(Nucleic Acid Res. 34:e128 (2006)).

An allele-specific primer/probe can be an oligonucleotide that isspecific for particular a polymorphism can be prepared using standardmethods. In some embodiments, allele-specific oligonucleotide probes canspecifically hybridize to a nucleic acid region that contains a geneticvariation. In some embodiments, hybridization conditions can be selectedsuch that a nucleic acid probe can specifically bind to the sequence ofinterest, for example, the variant nucleic acid sequence.

In some embodiments, allele-specific restriction digest analysis can beused to detect the existence of a polymorphic variant of a polymorphism,if alternate polymorphic variants of the polymorphism can result in thecreation or elimination of a restriction site. Allele-specificrestriction digests can be performed, for example, with the particularrestriction enzyme that can differentiate the alleles. In someembodiments, PCR can be used to amplify a region comprising thepolymorphic site, and restriction fragment length polymorphism analysiscan be conducted. In some embodiments, for sequence variants that do notalter a common restriction site, mutagenic primers can be designed thatcan introduce one or more restriction sites when the variant allele ispresent or when the wild type allele is present.

In some embodiments, fluorescence polarization template-directeddye-terminator incorporation (FP-TDI) can be used to determine which ofmultiple polymorphic variants of a polymorphism can be present in asubject. Unlike the use of allele-specific probes or primers, thismethod can employ primers that can terminate adjacent to a polymorphicsite, so that extension of the primer by a single nucleotide can resultin incorporation of a nucleotide complementary to the polymorphicvariant at the polymorphic site.

In some embodiments, DNA containing an amplified portion can bedot-blotted, using standard methods and the blot contacted with theoligonucleotide probe. The presence of specific hybridization of theprobe to the DNA can then be detected. The methods can includedetermining the genotype of a subject with respect to both copies of thepolymorphic site present in the genome, wherein if multiple polymorphicvariants exist at a site, this can be appropriately indicated byspecifying which variants are present in a subject. Any of the detectionmeans described herein can be used to determine the genotype of asubject with respect to one or both copies of the polymorphism presentin the subject's genome.

In some embodiments, a peptide nucleic acid (PNA) probe can be used inaddition to, or instead of, a nucleic acid probe in the methodsdescribed herein. A PNA can be a DNA mimic having a peptide-like,inorganic backbone, for example, N-(2-aminoethyl) glycine units with anorganic base (A, G, C, T or U) attached to the glycine nitrogen via amethylene carbonyl linker.

Nucleic acid sequence analysis can also be used to detect geneticvariations, for example, genetic variations can be detected bysequencing exons, introns, 5′ untranslated sequences, or 3′ untranslatedsequences. One or more methods of nucleic acid analysis that areavailable to those skilled in the art can be used to detect geneticvariations, including but not limited to, direct manual sequencing,automated fluorescent sequencing, single-stranded conformationpolymorphism assays (SSCP); clamped denaturing gel electrophoresis(CDGE); denaturing gradient gel electrophoresis (DGGE), two-dimensionalgel electrophoresis (2DGE or TDGE); conformational sensitive gelelectrophoresis (CSGE); denaturing high performance liquidchromatography (DHPLC), infrared matrix-assisted laserdesorption/ionization (IR-MALDI) mass spectrometry, mobility shiftanalysis, quantitative real-time PCR, restriction enzyme analysis,heteroduplex analysis; chemical mismatch cleavage (CMC), RNaseprotection assays, use of polypeptides that recognize nucleotidemismatches, allele-specific PCR, real-time pyrophosphate DNA sequencing,PCR amplification in combination with denaturing high performance liquidchromatography (dHPLC), and combinations of such methods.

Sequencing can be accomplished through classic Sanger sequencingmethods, which are known in the art. In some embodiments sequencing canbe performed using high-throughput sequencing methods some of whichallow detection of a sequenced nucleotide immediately after or upon itsincorporation into a growing strand, for example, detection of sequencein substantially real time or real time. In some cases, high throughputsequencing generates at least 1,000, at least 5,000, at least 10,000, atleast 20,000, at least 30,000, at least 40,000, at least 50,000, atleast 100,000 or at least 500,000 sequence reads per hour; with eachread being at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 120 or at least 150 bases per read (or500-1,000 bases per read for 454).

High-throughput sequencing methods can include but are not limited to,Massively Parallel Signature Sequencing (MPSS, Lynx Therapeutics),Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing,SOLiD sequencing, on semiconductor sequencing, DNA nanoball sequencing,Helioscope™ single molecule sequencing, Single Molecule SMRT™sequencing, Single Molecule real time (RNAP) sequencing, Nanopore DNAsequencing, and/or sequencing by hybridization, for example, anon-enzymatic method that uses a DNA microarray, or microfluidic Sangersequencing.

In some embodiments, high-throughput sequencing can involve the use oftechnology available by Helicos BioSciences Corporation (Cambridge,Mass.) such as the Single Molecule Sequencing by Synthesis (SMSS)method. SMSS is unique because it allows for sequencing the entire humangenome in up to 24 hours. This fast sequencing method also allows fordetection of a SNP/nucleotide in a sequence in substantially real timeor real time. Finally, SMSS is powerful because, like the MIPtechnology, it does not use a pre-amplification step prior tohybridization. SMSS does not use any amplification. SMSS is described inUS Publication Application Nos. 20060024711; 20060024678; 20060012793;20060012784; and 20050100932. In some embodiments, high-throughputsequencing involves the use of technology available by 454 LifeSciences, Inc. (a Roche company, Branford, Conn.) such as thePicoTiterPlate device which includes a fiber optic plate that transmitschemiluminescent signal generated by the sequencing reaction to berecorded by a CCD camera in the instrument. This use of fiber opticsallows for the detection of a minimum of 20 million base pairs in 4.5hours.

In some embodiments, PCR-amplified single-strand nucleic acid can behybridized to a primer and incubated with a polymerase, ATP sulfurylase,luciferase, apyrase, and the substrates luciferin and adenosine 5′phosphosulfate. Next, deoxynucleotide triphosphates corresponding to thebases A, C, G, and T (U) can be added sequentially. A base incorporationcan be accompanied by release of pyrophosphate, which can be convertedto ATP by sulfurylase, which can drive synthesis of oxyluciferin and therelease of visible light. Since pyrophosphate release can be equimolarwith the number of incorporated bases, the light given off can beproportional to the number of nucleotides adding in any one step. Theprocess can repeat until the entire sequence can be determined. In someembodiments, pyrosequencing can be utilized to analyze amplicons todetermine whether breakpoints are present. In some embodiments,pyrosequencing can map surrounding sequences as an internal qualitycontrol.

Pyrosequencing analysis methods are known in the art. Sequence analysiscan include a four-color sequencing by ligation scheme (degenerateligation), which involves hybridizing an anchor primer to one of fourpositions. Then an enzymatic ligation reaction of the anchor primer to apopulation of degenerate nonamers that are labeled with fluorescent dyescan be performed. At any given cycle, the population of nonamers that isused can be structured such that the identity of one of its positionscan be correlated with the identity of the fluorophore attached to thatnonamer. To the extent that the ligase discriminates for complementarilyat that queried position, the fluorescent signal can allow the inferenceof the identity of the base. After performing the ligation andfour-color imaging, the anchor primer: nonamer complexes can be strippedand a new cycle begins. Methods to image sequence information afterperforming ligation are known in the art.

In some embodiments, analysis by restriction enzyme digestion can beused to detect a particular genetic variation if the genetic variationresults in creation or elimination of one or more restriction sitesrelative to a reference sequence. In some embodiments, restrictionfragment length polymorphism (RFLP) analysis can be conducted, whereinthe digestion pattern of the relevant DNA fragment indicates thepresence or absence of the particular genetic variation in the nucleicacid sample.

In some embodiments, arrays of oligonucleotide probes that can becomplementary to target nucleic acid sequence segments from a subjectcan be used to identify genetic variations. In some embodiments, anarray of oligonucleotide probes comprises an oligonucleotide array, forexample, a microarray. In some embodiments, the present disclosurefeatures arrays that include a substrate having a plurality ofaddressable areas, and methods of using them. At least one area of theplurality includes a nucleic acid probe that binds specifically to asequence comprising a genetic variation, and can be used to detect theabsence or presence of the genetic variation, for example, one or moreSNPs, microsatellites, or CNVs, as described herein, to determine oridentify an allele or genotype. For example, the array can include oneor more nucleic acid probes that can be used to detect a geneticvariation associated with a gene and/or gene product. In someembodiments, the array can further comprise at least one area thatincludes a nucleic acid probe that can be used to specifically detectanother marker associated with a developmental disorder, for exampleAutism Spectrum Disorder, as described herein.

Microarray hybridization can be performed by hybridizing a nucleic acidof interest, for example, a nucleic acid encompassing a geneticvariation, with the array and detecting hybridization using nucleic acidprobes. In some embodiments, the nucleic acid of interest is amplifiedprior to hybridization. Hybridization and detecting can be carried outaccording to standard methods described in Published PCT Applications:WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186. For example,an array can be scanned to determine the position on the array to whichthe nucleic acid hybridizes. The hybridization data obtained from thescan can be, for example, in the form of fluorescence intensities as afunction of location on the array.

Arrays can be formed on substrates fabricated with materials such aspaper; glass; plastic, for example, polypropylene, nylon, orpolystyrene; polyacrylamide; nitrocellulose; silicon; optical fiber; orany other suitable solid or semisolid support; and can be configured ina planar, for example, glass plates or silicon chips); or threedimensional, for example, pins, fibers, beads, particles, microtiterwells, and capillaries, configuration.

Methods for generating arrays are known in the art and can include forexample; photolithographic methods (U.S. Pat. Nos. 5,143,854, 5,510,270and 5,527,681); mechanical methods, for example, directed-flow methods(U.S. Pat. No. 5,384,261); pin-based methods (U.S. Pat. No. 5,288,514);bead-based techniques (PCT US/93/04145); solid phase oligonucleotidesynthesis methods; or by other methods known to a person skilled in theart (see, e.g., Bier, F. F., et al. Adv Biochem Eng Biotechnol109:433-53 (2008); Hoheisel, J. D., Nat Rev Genet 7: 200-10 (2006); Fan,J. B., et al. Methods Enzymol 410:57-73 (2006); Raqoussis, J. & Elvidge,G., Expert Rev Mol Design 6: 145-52 (2006); Mockler, T. C., et al.Genomics 85: 1-15 (2005), and references cited therein, the entireteachings of each of which are incorporated by reference herein). Manyadditional descriptions of the preparation and use of oligonucleotidearrays for detection of polymorphisms can be found, for example, in U.S.Pat. Nos. 6,858,394, 6,429,027, 5,445,934, 5,700,637, 5,744,305,5,945,334, 6,054,270, 6,300,063, 6,733,977, 7,364,858, EP 619 321, andEP 373 203, the entire teachings of which are incorporated by referenceherein. Methods for array production, hybridization, and analysis arealso described in Snijders et al., Nat. Genetics 29:263-264 (2001);Klein et al., Proc. Natl. Acad. Sci. USA 96:4494-4499 (1999); Albertsonet al., Breast Cancer Research and Treatment 78:289-298 (2003); andSnijders et al., “BAC microarray based comparative genomichybridization,” in: Zhao et al. (eds), Bacterial Artificial Chromosomes:Methods and Protocols, Methods in Molecular Biology, Humana Press, 2002.

In some embodiments, oligonucleotide probes forming an array can beattached to a substrate by any number of techniques, including, but notlimited to, in situ synthesis, for example, high-density oligonucleotidearrays, using photolithographic techniques; spotting/printing a mediumto low density on glass, nylon, or nitrocellulose; by masking; and bydot-blotting on a nylon or nitrocellulose hybridization membrane. Insome embodiments, oligonucleotides can be immobilized via a linker,including but not limited to, by covalent, ionic, or physical linkage.Linkers for immobilizing nucleic acids and polypeptides, includingreversible or cleavable linkers, are known in the art (U.S. Pat. No.5,451,683 and WO98/20019). In some embodiments, oligonucleotides can benon-covalently immobilized on a substrate by hybridization to anchors,by means of magnetic beads, or in a fluid phase, for example, in wellsor capillaries.

An array can comprise oligonucleotide hybridization probes capable ofspecifically hybridizing to different genetic variations. In someembodiments, oligonucleotide arrays can comprise a plurality ofdifferent oligonucleotide probes coupled to a surface of a substrate indifferent known locations. In some embodiments, oligonucleotide probescan exhibit differential or selective binding to polymorphic sites, andcan be readily designed by one of ordinary skill in the art, forexample, an oligonucleotide that is perfectly complementary to asequence that encompasses a polymorphic site, for example, a sequencethat includes the polymorphic site, within it, or at one end, canhybridize preferentially to a nucleic acid comprising that sequence, asopposed to a nucleic acid comprising an alternate polymorphic variant.

In some embodiments, arrays can include multiple detection blocks, forexample, multiple groups of probes designed for detection of particularpolymorphisms. In some embodiments, these arrays can be used to analyzemultiple different polymorphisms. In some embodiments, detection blockscan be grouped within a single array or in multiple, separate arrays,wherein varying conditions, for example, conditions optimized forparticular polymorphisms, can be used during hybridization. Generaldescriptions of using oligonucleotide arrays for detection ofpolymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and5,837,832. In addition to oligonucleotide arrays, cDNA arrays can beused similarly in certain embodiments.

The methods described herein can include but are not limited toproviding an array as described herein; contacting the array with anucleic acid sample, and detecting binding of a nucleic acid from thenucleic acid sample to the array. In some embodiments, the method cancomprise amplifying nucleic acid from the nucleic acid sample, forexample, a region associated with a developmental disorder or a regionthat includes another region associated with a developmental disorder.In some embodiments, the methods described herein can include using anarray that can identify differential expression patterns or copy numbersof one or more genes in nucleic acid samples from control and affectedindividuals. For example, arrays of probes to a marker described hereincan be used to identify genetic variations between DNA from an affectedsubject, and control DNA obtained from an individual that does not havea developmental disorder. Since the nucleotides on the array can containsequence tags, their positions on the array can be accurately knownrelative to the genomic sequence

In some embodiments, it can be desirable to employ methods that candetect the presence of multiple genetic variations, for example,polymorphic variants at a plurality of polymorphic sites, in parallel orsubstantially simultaneously. In some embodiments, these methods cancomprise oligonucleotide arrays and other methods, including methods inwhich reactions, for example, amplification and hybridization, can beperformed in individual vessels, for example, within individual wells ofa multi-well plate or other vessel.

Determining the identity of a genetic variation can also include orconsist of reviewing a subject's medical history, where the medicalhistory includes information regarding the identity, copy number,presence or absence of one or more alleles or SNPs in the subject, e.g.,results of a genetic test.

In some embodiments extended runs of homozygosity (ROH) may be useful tomap recessive disease genes in outbred populations. Furthermore, even incomplex disorders, a high number of affected individuals may have thesame haplotype in the region surrounding a disease mutation. Therefore,a rare pathogenic variant and surrounding haplotype can be enriched infrequency in a group of affected individuals compared with the haplotypefrequency in a cohort of unaffected controls. Homozygous haplotypes (HH)that are shared by multiple affected individuals can be important forthe discovery of recessive disease genes in complex disorders such asASD. In some embodiments, the traditional homozygosity mapping methodcan be extended by analysing the haplotype within shared ROH regions toidentify homozygous segments of identical haplotype that are presentuniquely or at a higher frequency in ASD probands compared to parentalcontrols. Such regions are termed risk homozygous haplotypes (rHH),which may contain low-frequency recessive variants that contribute toASD risk in a subset of ASD patients.

Genetic variations can also be identified using any of a number ofmethods well known in the art. For example, genetic variations availablein public databases, which can be searched using methods and customalgorithms or algorithms known in the art, can be used. In someembodiments, a reference sequence can be from, for example, the humandraft genome sequence, publicly available in various databases, or asequence deposited in a database such as GenBank.

Any of the polynucleotides described, including polynucleotidescomprising a genetic variation, can be made synthetically using methodsknown in the art.

Methods of Detecting CNVs

Detection of genetic variations, specifically CNVs, can be accomplishedby one or more suitable techniques described herein. Generally,techniques that can selectively determine whether a particularchromosomal segment is present or absent in an individual can be usedfor genotyping CNVs. Identification of novel copy number variations canbe done by methods for assessing genomic copy number changes.

In some embodiments, methods include but are not limited to, methodsthat can quantitatively estimate the number of copies of a particulargenomic segment, but can also include methods that indicate whether aparticular segment is present in a nucleic acid sample or not. In someembodiments, the technique to be used can quantify the amount of segmentpresent, for example, determining whether a DNA segment is deleted,duplicated, or triplicated in subject, for example, Fluorescent In SituHybridization (FISH) techniques, and other methods described herein. Insome embodiments, methods include detection of copy number variationfrom array intensity and sequencing read depth using a stepwise Bayesianmodel (Zhang Z. D., et al. BMC Bioinformatics. 2010 Oct. 31; 11:539). Insome embodiments, methods include detecting copy number variations usingshotgun sequencing, CNV-seq (Xie C., et al. BMC Bioinformatics. 2009Mar. 6; 10:80). In some embodiments, methods include analyzingnext-generation sequencing (NGS) data for CNV detection using any one ofseveral algorithms developed for each of the four broad methods for CNVdetection using NGS, namely the depth of coverage (DOC), read-pair (RP),split-read (SR) and assembly-based (AS) methods. (Teo S. M., et al.Bioinformatics. 2012 Aug. 31). In some embodiments, methods includecombining coverage with map information for the identification ofdeletions and duplications in targeted sequence data (Nord A. S., et al.BMC Genomics. 2011 Apr. 12; 12:184).

In some embodiments, other genotyping technologies can be used fordetection of CNVs, including but not limited to, karyotype analysis,Molecular Inversion Probe array technology, for example, Affymetrix SNPArray 6.0, and BeadArray Technologies, for example, Illumina GoldenGateand Infinium assays, as can other platforms such as NimbleGen HD2.1 orHD4.2, High-Definition Comparative Genomic Hybridization (CGH) arrays(Agilent Technologies), tiling array technology (Affymetrix), multiplexligation-dependent probe amplification (MLPA), Invader assay,fluorescence in situ hybridization, and, in one embodiment, ArrayComparative Genomic Hybridization (aCGH) methods. As described herein,karyotype analysis can be a method to determine the content andstructure of chromosomes in a nucleic acid sample. In some embodiments,karyotyping can be used, in lieu of aCGH, to detect translocations,which can be copy number neutral, and, therefore, not detectable byaCGH. Information about amplitude of particular probes, which can berepresentative of particular alleles, can provide quantitative dosageinformation for the particular allele, and by consequence, dosageinformation about the CNV in question, since the marker can be selectedas a marker representative of the CNV and can be located within the CNV.In some embodiments, if the CNV is a deletion, the absence of particularmarker allele is representative of the deletion. In some embodiments, ifthe CNV is a duplication or a higher order copy number variation, thesignal intensity representative of the allele correlating with the CNVcan represent the copy number. A summary of methodologies commonly usedis provided in Perkel (Perkel J. Nature Methods 5:447-453 (2008)).

PCR assays can be utilized to detect CNVs and can provide an alternativeto array analysis. In particular, PCR assays can enable detection ofprecise boundaries of gene/chromosome variants, at the molecular level,and which boundaries are identical in different individuals. PCR assayscan be based on the amplification of a junction fragment present only inindividuals that carry a deletion. This assay can convert the detectionof a loss by array CGH to one of a gain by PCR.

Examples of PCR techniques that can be used in the present disclosureinclude, but are not limited to quantitative PCR, real-time quantitativePCR (qPCR), quantitative fluorescent PCR (QF-PCR), multiplex fluorescentPCR (MF-PCR), real time PCR (RT-PCR), single cell PCR,PCR-RFLP/RT-PCR-RFLP, hot start PCR and Nested PCR. Other suitableamplification methods include the ligase chain reaction (LCR), ligationmediated PCR (LM-PCR), degenerate oligonucleotide probe PCR (DOP-PCR),transcription amplification, self-sustained sequence replication,selective amplification of target polynucleotide sequences, consensussequence primed polymerase chain reaction (CP-PCR), arbitrarily primedpolymerase chain reaction (AP-PCR) and nucleic acid based sequenceamplification (NABSA).

Alternative methods for the simultaneous interrogation of multipleregions include quantitative multiplex PCR of short fluorescentfragments (QMPSF), multiplex amplifiable probe hybridization (MAPH) andmultiplex ligation-dependent probe amplification (MLPA), in whichcopy-number differences for up to 40 regions can be scored in oneexperiment. Another approach can be to specifically target regions thatharbor known segmental duplications, which are often sites ofcopy-number variation. By targeting the variable nucleotides between twocopies of a segmental duplication (called paralogous sequence variants)using a SNP-genotyping method that provides independent fluorescenceintensities for the two alleles, it is possible to detect an increase inintensity of one allele compared with the other.

In some embodiments, the amplified piece of DNA can be bound to beadsusing the sequencing element of the nucleic acid tag under conditionsthat favor a single amplified piece of DNA molecule to bind a differentbead and amplification occurs on each bead. In some embodiments, suchamplification can occur by PCR. Each bead can be placed in a separatewell, which can be a picoliter-sized well. In some embodiments, eachbead is captured within a droplet of aPCR-reaction-mixture-in-oil-emulsion and PCR amplification occurs withineach droplet. The amplification on the bead results in each beadcarrying at least one million, at least 5 million, or at least 10million copies of the single amplified piece of DNA molecule.

In embodiments where PCR occurs in oil-emulsion mixtures, the emulsiondroplets are broken, the DNA is denatured and the beads carryingsingle-stranded nucleic acids clones are deposited into a well, such asa picoliter-sized well, for further analysis according to the methodsdescribed herein. These amplification methods allow for the analysis ofgenomic DNA regions. Methods for using bead amplification followed byfiber optics detection are described in Margulies et al. 2005, Nature.15; 437(7057):376-80, and as well as in US Publication Application Nos.20020012930; 20030068629; 20030100102; 20030148344; 20040248161;20050079510, 20050124022; and 20060078909.

Another variation on the array-based approach can be to use thehybridization signal intensities that are obtained from theoligonucleotides employed on Affymetrix SNP arrays or in Illumina BeadArrays. Here hybridization intensities are compared with average valuesthat are derived from controls, such that deviations from these averagesindicate a change in copy number. As well as providing information aboutcopy number, SNP arrays have the added advantage of providing genotypeinformation. For example, they can reveal loss of heterozygosity, whichcould provide supporting evidence for the presence of a deletion, ormight indicate segmental uniparental disomy (which can recapitulate theeffects of structural variation in some genomic regions—Prader-Willi andAngelman syndromes, for example).

Many of the basic procedures followed in microarray-based genomeprofiling are similar, if not identical, to those followed in expressionprofiling and SNP analysis, including the use of specialized microarrayequipment and data-analysis tools. Since microarray-based expressionprofiling has been well established in the last decade, much can belearned from the technical advances made in this area. Examples of theuse of microarrays in nucleic acid analysis that can be used aredescribed in U.S. Pat. Nos. 6,300,063, 5,837,832, 6,969,589, 6,040,138,6,858,412, U.S. application Ser. No. 08/529,115, U.S. application Ser.No. 10/272,384, U.S. application Ser. No. 10/045,575, U.S. applicationSer. No. 10/264,571 and U.S. application Ser. No. 10/264,574. It shouldbe noted that there are also distinct differences such as target andprobe complexity, stability of DNA over RNA, the presence of repetitiveDNA and the need to identify single copy number alterations in genomeprofiling.

In some embodiments, the genetic variations detected comprise CNVs andcan be detected using array CGH. In some embodiments, array CGH can bebeen implemented using a wide variety of techniques. The initialapproaches used arrays produced from large-insert genomic clones such asbacterial artificial chromosomes (BACs). Producing sufficient BAC DNA ofadequate purity to make arrays is arduous, so several techniques toamplify small amounts of starting material have been employed. Thesetechniques include ligation-mediated PCR (Snijders et al, Nat. Genet.29:263-64), degenerate primer PCR using one or several sets of primers,and rolling circle amplification. BAC arrays that provide completegenome tiling paths are also available. Arrays made from less complexnucleic acids such as cDNAs, selected PCR products, and oligonucleotidescan also be used. Although most CGH procedures employ hybridization withtotal genomic DNA, it is possible to use reduced complexityrepresentations of the genome produced by PCR techniques. Computationalanalysis of the genome sequence can be used to design array elementscomplementary to the sequences contained in the representation. VariousSNP genotyping platforms, some of which use reduced complexity genomicrepresentations, can be useful for their ability to determine both DNAcopy number and allelic content across the genome. In some embodiments,small amounts of genomic DNA can be amplified with a variety of wholegenome or whole exome amplification methods prior to CGH analysis of thenucleic acid sample. A “whole exome,” as used herein, includes s exonsthroughout the whole genome that are expressed in genes. Since exonselection has tissue and cell type specificity, these positions may bedifferent in the various cell types resulting from a splice variant oralternative splicing. A “whole genome,” as used herein, includes theentire genetic code of a genome.

The different basic approaches to array CGH provide different levels ofperformance, so some are more suitable for particular applications thanothers. The factors that determine performance include the magnitudes ofthe copy number changes, their genomic extents, the state andcomposition of the specimen, how much material is available foranalysis, and how the results of the analysis can be used. Manyapplications use reliable detection of copy number changes of much lessthan 50%, a more stringent requirement than for other microarraytechnologies. Note that technical details are extremely important anddifferent implementations of methods using the same array CGH approachcan yield different levels of performance. Various CGH methods are knownin the art and are equally applicable to one or more methods of thepresent disclosure. For example, CGH methods are disclosed in U.S. Pat.Nos. 7,030,231; 7,011,949; 7,014,997; 6,977,148; 6,951,761; and6,916,621, the disclosure from each of which is incorporated byreference herein in its entirety.

The data provided by array CGH are quantitative measures of DNA sequencedosage. Array CGH provides high-resolution estimates of copy numberaberrations, and can be performed efficiently on many nucleic acidsamples. The advent of array CGH technology makes it possible to monitorDNA copy number changes on a genomic scale and many projects have beenlaunched for studying the genome in specific diseases.

In some embodiments, whole genome array-based comparative genomehybridization (array CGH) analysis, or array CGH on a subset of genomicregions, can be used to efficiently interrogate human genomes forgenomic imbalances at multiple loci within a single assay. Thedevelopment of comparative genomic hybridization (CGH) (Kallioniemi etal, 1992, Science 258: 818-21) provided the first efficient approach toscanning entire genomes for variations in DNA copy number. Theimportance of normal copy number variation involving large segments ofDNA has been unappreciated. Array CGH is a breakthrough technique inhuman genetics, which is attracting interest from clinicians working infields as diverse as cancer and IVF (In Vitro Fertilization). The use ofCGH microarrays in the clinic holds great promise for identifyingregions of genomic imbalance associated with disease. Advances fromidentifying chromosomal critical regions associated with specificphenotypes to identifying the specific dosage sensitive genes can leadto therapeutic opportunities of benefit to patients. Array CGH is aspecific, sensitive and rapid technique that can enable the screening ofthe whole genome in a single test. It can facilitate and accelerate thescreening process in human genetics and is expected to have a profoundimpact on the screening and counseling of patients with geneticdisorders. It is now possible to identify the exact location on thechromosome where an aberration has occurred and it is possible to mapthese changes directly onto the genomic sequence.

An array CGH approach provides a robust method for carrying out agenome-wide scan to find novel copy number variants (CNVs). The arrayCGH methods can use labeled fragments from a genome of interest, whichcan be competitively hybridized with a second differentially labeledgenome to arrays that are spotted with cloned DNA fragments, revealingcopy-number differences between the two genomes. Genomic clones (forexample, BACs), cDNAs, PCR products and oligonucleotides, can all beused as array targets. The use of array CGH with BACs was one of theearliest employed methods and is popular, owing to the extensivecoverage of the genome it provides, the availability of reliable mappingdata and ready access to clones. The last of these factors is importantboth for the array experiments themselves, and for confirmatory FISHexperiments.

In a typical CGH measurement, total genomic DNA is isolated from controland reference subjects, differentially labeled, and hybridized to arepresentation of the genome that allows the binding of sequences atdifferent genomic locations to be distinguished. More than two genomescan be compared simultaneously with suitable labels. Hybridization ofhighly repetitive sequences is typically suppressed by the inclusion ofunlabeled Cot-1 DNA in the reaction. In some embodiments of array CGH,it is beneficial to mechanically shear the genomic DNA in a nucleic acidsample, for example, with sonication, prior to its labeling andhybridization step. In another embodiment, array CGH may be performedwithout use of Cot-1 DNA or a sonication step in the preparation of thegenomic DNA in a nucleic acid sample. The relative hybridizationintensity of the test and reference signals at a given location can beproportional to the relative copy number of those sequences in the testand reference genomes. If the reference genome is normal then increasesand decreases in signal intensity ratios directly indicate DNA copynumber variation within the genome of the test cells. Data are typicallynormalized so that the modal ratio for the genome is set to somestandard value, typically 1.0 on a linear scale or 0.0 on a logarithmicscale. Additional measurements such as FISH or flow cytometry can beused to determine the actual copy number associated with a ratio level.

In some embodiments, an array CGH procedure can include the followingsteps. First, large-insert clones, for example, BACs can be obtainedfrom a supplier of clone libraries. Then, small amounts of clone DNA canbe amplified, for example, by degenerate oligonucleotide-primed (DOP)PCR or ligation-mediated PCR in order to obtain sufficient quantitiesneeded for spotting. Next, PCR products can be spotted onto glass slidesusing, for example, microarray robots equipped with high-precisionprinting pins. Depending on the number of clones to be spotted and thespace available on the microarray slide, clones can either be spottedonce per array or in replicate. Repeated spotting of the same clone onan array can increase precision of the measurements if the spotintensities are averaged, and allows for a detailed statistical analysisof the quality of the experiments. Subject and control DNAs can belabeled, for example, with either Cy3 or Cy5-dUTP using random primingand can be subsequently hybridized onto the microarray in a solutioncontaining an excess of Cotl-DNA to block repetitive sequences.Hybridizations can either be performed manually under a coverslip, in agasket with gentle rocking or, automatically using commerciallyavailable hybridization stations. These automated hybridization stationscan allow for an active hybridization process, thereby improving thereproducibility as well as reducing the actual hybridization time, whichincreases throughput. The hybridized DNAs can detected through the twodifferent fluorochromes using standard microarray scanning equipmentwith either a scanning confocal laser or a charge coupled device (CCD)camera-based reader, followed by spot identification using commerciallyor freely available software packages.

The use of CGH with arrays that comprise long oligonucleotides (60-100bp) can improve the detection resolution (in some embodiments, as smallas ˜3-5 kb sized CNVs on arrays designed for interrogation of humanwhole genomes) over that achieved using BACs (limited to 50-100 kb orlarger sized CNVs due to the large size of BAC clones). In someembodiments, the resolution of oligonucleotide CGH arrays is achievedvia in situ synthesis of 1-2 million unique features/probes permicroarray, which can include microarrays available from Roche NimbleGenand Agilent Technologies. In addition to array CGH methods for copynumber detecton, other embodiments for partial or whole genome analysisof CNVs within a genome include, but are not limited to, use of SNPgenotyping microarrays and sequencing methods.

Another method for copy number detection that uses oligonucleotides canbe representational oligonucleotide microarray analysis (ROMA). It issimilar to that applied in the use of BAC and CGH arrays, but toincrease the signal-to-noise ratio, the ‘complexity’ of the input DNA isreduced by a method called representation or whole-genome sampling. Herethe DNA that is to be hybridized to the array can be treated byrestriction digestion and then ligated to adapters, which results in thePCR-based amplification of fragments in a specific size-range. As aresult, the amplified DNA can make up a fraction of the entire genomicsequence—that is, it is a representation of the input DNA that hassignificantly reduced complexity, which can lead to a reduction inbackground noise. Other suitable methods available to the skilled personcan also be used, and are within scope of the present disclosure.

A comparison of one or more genomes relative to one or more othergenomes with array CGH, or a variety of other CNV detection methods, canreveal the set of CNVs between two genomes, between one genome incomparison to multiple genomes, or between one set of genomes incomparison to another set of genomes. In some embodiments, an array CGHexperiment can be performed by hybrizing a single test genome against apooled nucleic acid sample of two or more genomes, which can result inminimizing the detection of higher frequency variants in the experiment.In some embodiments, a test genome can be hybridized alone (i.e.,one-color detetion) to a microarray, for example, using array CGH or SNPgenotyping methods, and the comparison step to one or more referencegenomes can be performed in silico to reveal the set of CNVs in the testgenome relative to the one or more reference genomes. In one preferredembodiment, a single test genome is compared to a single referencegenome in a 2-color experiment wherein both genomes are cohybridized tothe microarray.

Array CGH can be used to identify genes that are causative or associatedwith a particular phenotype, condition, or disease by comparing the setof CNVs found in the affected cohort to the set of CNVs found in anunaffected cohort. An unaffected cohort may consist of any individualunaffected by the phenotype, condition, or disease of interest, but inone preferred embodiment is comprised of individuals or subjects thatare apparently healthy (normal). Methods employed for such analyses aredescribed in U.S. Pat. Nos. 7,702,468 and 7,957,913. In some embodimentsof CNV comparison methods, candidate genes that are causative orassociated (i.e., potentially serving as a biomarker) with a phenotype,condition, or disease will be identified by CNVs that occur in theaffected cohort but not in the unaffected cohort. In some embodiments ofCNV comparison methods, candidate genes that are causative or associated(i.e., potentially serving as a biomarker) with a phenotype, condition,or disease will be identified by CNVs that occur at a statisticallysignificant higher frequency in the affected cohort as compared theirfrequency in the unaffected cohort. Thus, CNVs preferentially detectedin the affected cohort as compared to the unaffected cohort can serve asbeacons of genes that are causative or associated with a particularphenotype, condition, or disease. In some embodiments, CNV detection andcomparison methods can result in direct identification of the gene thatis causative or associated with phenotype, condition, or disease if theCNVs are found to overlap with or encompass the gene(s). In someembodiments, CNV detection and comparison methods can result inidentification of regulatory regions of the genome (e.g., promoters,enhancers, transcription factor binding sites) that regulate theexpression of one or more genes that are causative or associated withthe phenotype, condition, or disease of interest.

Due to the large amount of genetic variation between any two genomes, ortwo sets (cohorts) of genomes, being compared, one preferred embodimentis to reduce the genetic variation search space by interrogating onlyCNVs, as opposed to the full set of genetic variants that can beidentified in an individual's genome or exome. The set of CNVs thatoccur only, or at a statistically higher frequency, in the affectedcohort as compared to the unaffected cohort can then be furtherinvestigated in targeted sequencing experiments to reveal the full setof genetic variants (of any size or type) that are causative orassociated (i.e., potentially serving as a biomarker) with a phenotype,condition, or disease. It can be appreciated to those skilled in the artthat the targeted sequencing experiments are performed in both theaffected and unaffected cohorts in order to identify the geneticvariants (e.g., SNVs and indels) that occur only, or at a statisticallysignificant higher frequency, in the affected individual or cohort ascompared to the unaffected cohort.

When investigating a particular phenotype, condition, or disease, suchas ASD, it can be appreciated by those skilled in the art that thenumber of ASD candidate genes (or regulatory sequences) identified viaCNV (or other variant types) detection methods may increase or decreasewhen additional ASD cohorts are analyzed. Similarly, the number of ASDcandidate genes (or regulatory sequences), for example, identified viaCNV (or other variant types) detection methods may increase or decreasewhen additional unaffected cohorts are used to interpret the affectedcohort CNVs (or other variat types). For very rare CNVs (e.g., <0.1%frequency in the general population), only a single case may be observedin a given ASD cohort (e.g., 100 cases) but further statisticalsignificance or evidence for the gene (or regulatory sequence/locus inthe genome) can be established by: 1) CNV analysis of additional ASDcohorts, 2) CNV analysis of additional Normal cohorts, 3) targeted genesequencing of both ASD and Normal cohorts, and/or 4) functionalcharacterization of the ASD candidate gene (e.g., in silico analysis ofthe predicted impact of the candidate mutation on the gene product, RNAiknockdown experiments, biochemical assays on ASD patient tissue, geneexpression analysis of disease-relevant tissues or of inducedpluripotent stem cells (iPSCs) created from the ASD patient(s) harboringthe candidate ASD-causing genetic variant).

A candidate gene may validate as causative of the phenotype, condition,or disease (e.g., ASD), which may, for example, be confirmed viamechansism of action experiments, or it may serve as a biomarker of thephenotype, condition, or disease. Thus, in the example of ASD, in someembodiments, the ASD-specific gene (or regulatory sequence/locus) may bea biomarker of age-of-onset for ASD and disease severity, and thus havediagnostic utility for monitoring patients known to be at risk for ASDor as a general screening test in the population for early diagnosis ofthe disease. In some embodiments, the ASD-specific gene/biomarker may bean indicator of drug response (e.g., a particular subtype of ASD mayrespond best to a therapeutic targeting a particular phenotype,causative gene, or other gene in the same pathway as the causative gene)and thus have utility during drug development in clinical trials. Forexample, clinical trials for a therapeutic that targets a ASD geneticsubtype comprising only 10% of all patients exhibiting symptoms of ASD,can be designed to comprise only those 10% of patients with a specificgenotype(s) in order to reduce the time and cost of such clinical trials(e.g., smaller number of patients in the clinical trial). It can beappreciated by those skilled in the art that such patient stratificationmethods (i.e., specific genotypes correlated with the disease or drugresponse) can be employed not only for targeted therapeutics, but ingeneral for any drug that is approved or in development (i.e., themechanism of action may or may not be known). For example, drugs indevelopment or approved to treat, for example, cancer, may have utilityin being repurposed to treat ASD. Such patient stratification methodscan also be utilized to develop a companion diagnostic test (e.g.,comprising the specific genes/genotypes found in patients that areindicative of drug response) for a particular drug, either concurrentlyduring the clinical trials for the drug or after drug approval (e.g., asa new indication or for the physician to use in guiding medicaldecisions for the patient).

Further neurodevelopmental and/or links to ASD pathology can beestablished via pathway analysis of the genes, which may take intoconsideration binding interactions (e.g., via yeast 2-hybrid screen) andmolecular events (e.g., kinase activity or other enzymatic processes) ifsuch information is available for the gene(s) of interest (i.e.,specified in the analysis). Both commercial (e.g., Ingenuity's IPAsoftware and Thomson Reuter's GeneGo software) and open source software(e.g., String: string-db.org/) are available for such analyses. Toassess connections to established ASD biology, analyses can be performedfor the set of candidate ASD genes independently or against knowncausative ASD genes (e.g., FMR1, MECP2, and contactins such as CNTN4)singly or as a group. In some embodiments, ASD candidate genes can bedistributed into one or more of several categories: 1) linked to a knowncausative ASD gene (e.g., binding partner) or a novel family member of aknown ASD gene, 2) apoptosis pathway, 3) cell signaling (e.g., smallGTPases, Wnt), 4) metabolism defects (e.g., amino acids,purines/pyrimidines), mitochondrial dysfunction, 5) neuroprotectivefactors, 6) neurotransmitter signaling, 7) synapse formation/function,8) ubiquitin/proteasome pathway, 9) neuropsychiatric genes, some ofwhich are known drug targets, and 10) other (e.g., established role inother diseases with no obvious neurodevelopmental biology, such ascancer) or unknown gene function (e.g., limited or no gene informationpresently annotated for the ASD-specific gene).

A method of screening a subject for a disease or disorder can compriseassaying a nucleic acid sample from the subject to detect sequenceinformation for more than one genetic locus and comparing the sequenceinformation to a panel of nucleic acid biomarkers and screening thesubject for the presence or absence of the disease or disorder if one ormore of low frequency biomarkers in the panel are present in thesequence information.

The panel can comprise at least one nucleic acid biomarker for each ofthe more than one genetic loci. For example, the panel can comprise 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200 or more nucleic acid biomarkers for each of the more than onegenetic locus. In some embodiments, the panel can comprise from about2-1000 nucleic acid biomarkers. For example, the panel can comprise fromabout 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2-300, 2-200, 2-100,25-900, 25-800, 25-700, 25-600, 25-500, 25-400, 25-300, 25-200, 25-100,100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300,100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400,200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400,400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000,500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700,700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 nucleic acidbiomarkers.

The panel can comprise at least 2 low frequency biomarkers. For example,the panel can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 3,14, 15, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 500, or 1000 or more lowfrequency biomarkers. In some embodiments, the panel can comprise fromabout 2-1000 low frequency biomarkers. For example, the panel cancomprise from about 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2-300,2-200, 2-100, 25-900, 25-800, 25-700, 25-600, 25-500, 25-400, 25-300,25-200, 25-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500,100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600,200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600,300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500,500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900,600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or900-1000 1000 low frequency biomarkers. In some embodiments, a lowfrequency biomarker can occur at a frequency of 0.1% or less in apopulation of subjects without a diagnosis of the disease or disorder.For example, a low frequency biomarker can occur at a frequency of0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, or 0.00001% orless in a population of subjects without a diagnosis of the disease ordisorder. In some embodiments, a low frequency biomarker can occur at afrequency from about 0.00001%-0.1% in a population of subjects without adiagnosis of the disease or disorder. For example, a low frequencybiomarker can occur at a frequency of from about 0.00001%-0.00005%,0.00001%-0.0001%, 0.00001%-0.0005%, 0.00001%-0.001%, 0.00001%-0.005%,0.00001%-0.01%, 0.00001%-0.05%, 0.00005%-0.0001%, 0.00005%-0.0005%,0.00005%-0.001%, 0.00005%-0.005%, 0.00005%-0.01%, 0.00005%-0.05%,0.00005%-0.1%, 0.0001%-0.0005%, 0.0001%-0.001%, 0.0001%-0.005%,0.0001%-0.01%, 0.0001%-0.05%, 0.0001%-0.1%, 0.0005%-0.001%,0.0005%-0.005%, 0.0005%-0.01%, 0.0005%-0.05%, 0.0005%-0.1%,0.001%-0.005%, 0.001%-0.01%, 0.001%-0.05%, 0.001%-0.1%, 0.005%-0.01%,0.005%-0.05%, 0.005%-0.1%, 0.01%-0.05%, 0.01%-0.1%, or 0.05%-0.1% in apopulation of subjects without a diagnosis of the disease or disorder

In some embodiments, the presence or absence of the disease or disorderin the subject can be determined with at least 50% confidence. Forexample, the presence or absence of the disease or disorder in thesubject can be determined with at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% confidence. In some embodiments,the presence or absence of the disease or disorder in the subject can bedetermined with a 50%-100% confidence. For example, the presence orabsence of the disease or disorder in the subject can be determined witha 60%-100%, 70%-100%, 80%-100%, 90%-100%, 50%-90%, 50%-80%, 50%-70%,50%-60%, 60%-90%, 60%-80%, 60%-70%, 70%-90%, 70%-80%, or 80%-90%. In oneembodiment, ASD candidate CNVs and genes or regulatory loci associatedwith these CNVs can be determined or identified by comparing geneticdata from a cohort of normal individuals to that of an individual or acohort of individuals known to have, or be susceptible to adevelopmental disorder such as ASD.

In one embodiment, ASD candidate CNV-subregions and genes associatedwith these regions can be determined or identified by comparing geneticdata from a cohort of normal individuals, such as a pre-existingdatabase of CNVs found in normal individuals termed the Normal VariationEngine (NVE), to that of a cohort of individual known to have, or besusceptible to a developmental disorder such as ASD.

In some embodiments, a nucleic acid sample from one individual ornucleic acid samples from a pool of 2 or more individuals without ASDcan serve as as the reference nucleic acid sample(s) and the nucleicacid sample from an individual known to have ASD or being tested todetermine if they have ASD can serve as the test nucleic acid sample. Inone preferred embodiment, the reference and test nucleic acid samplesare sex-matched and co-hybridized on the CGH array. For example,reference nucleic acid samples can be labeled with a fluorophore such asCy5, using methods described herein, and test subject nucleic acidsamples can be labeled with a different fluorophore, such as Cy3. Afterlabeling, nucleic acid samples can be combined and can be co-hybridizedto a microarray and analyzed using any of the methods described herein,such as aCGH. Arrays can then be scanned and the data can be analyzedwith software. Genetic alterations, such as CNVs, can be called usingany of the methods described herein. A list of the genetic alterations,such as CNVs, can be generated for one or more test subjects and/or forone or more reference subjects. Such lists of CNVs can be used togenerate a master list of non-redundant CNVs and/or CNV-subregions foreach type of cohort. In one embodiment, a cohort of test nucleic acidsamples, such as individuals known to have or suspected to have ASD, canbe cohybridized with an identical sex-matched reference individual orsex-matched pool of reference individuals to generate a list ofredundant or non-redudant CNVs. Such lists can be based on the presenceor absence of one or more CNVs and/or CNV subregions present inindividuals within the cohort. In this manner, a master list can containa number of distinct CNVs and/or CNV-subregions, some of which areuniquely present in a single individual and some of which are present inmultiple individuals.

In some embodiments, CNVs and/or CNV-subregions of interest can beobtained by annotation of each CNV and/or CNV-subregion with relevantinformation, such as overlap with known genes and/or exons exons orintergenic regulatory regions such as transcription factor bindingsites. In some embodiments, CNVs and/or CNV-subregions of interest canbe obtained by calculating the OR for a CNV and/or CNV-subregionaccording to the following formula: OR=(ASD/((#individuals in ASDcohort)−ASD))/(NVE/((#individuals in NVE cohort)−NVE)), where:ASD=number of ASD individuals with a CNV-subregion of interest andNVE=number of NVE subjects with the CNV-subregion of interest. If NVE=0,it can be set to 1 to avoid dealing with infinities in cases where noCNVs are seen in the NVE. In some embodiments, a set of publiclyavailable CNVs (e.g., the Database of Genomic Variants) can be used asthe Normal cohort for comparison to the affected cohort CNVs. In anotherembodiment, the set of Normal cohort CNVs may comprise a privatedatabase generated by the same CNV detection method, such as array CGH,or by a plurality of CNV detection methods that include, but are notlimited to, array CGH, SNP genotyping arrays, custom CGH arrays, customgenotyping arrays, exome sequencing, whole genome sequencing, targetedsequencing, FISH, q-PCR, or MLPA.

The number of individuals in any given cohort can be at least about 10,50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, 7500,10,000, 100,000, or more. In some embodiments, the number of individualsin any given cohort can be from 25-900, 25-800, 25-700, 25-600, 25-500,25-400, 25-300, 25-200, 25-100, 100-1000, 100-900, 100-800, 100-700,100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800,200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800,300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700,400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600,600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700-800,800-1000, 800-900, or 900-1000.

In some embodiments, a method of determining relevance or statisticalsignificance of a genetic variant in a human subject to a disease or acondition associated with a genotype comprising screening a genome of ahuman subject with the disease or condition, such as by arrayComparative Genomic Hybridization, sequencing, or SNP genotyping, toprovide information on one or more genetic variants, such as those inTables 1 and 2. The method can further comprise comparing, such as via acomputer, information of said one or more genetic variants from thegenome of said subject to a compilation of data comprising frequenciesof genetic variants in at least 100 normal human subjects, such as thosewithout the disease or condition. The method can further comprisedetermining a statistical significance or relevance of said one or moregenetic variants from said comparison to the condition or disease ordetermining whether a genetic variant is present in said human subjectbut not present in said compilation of data from said comparison, or analgorithm can be used to call or identify significant geneticvariations, such as a genetic variation whose median log 2 ratio isabove or below a computed value. A computer can comprise computerexecutable logic that provides instructions for executing saidcomparison.

Different categories for CNVs of interest can be defined. In someembodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions do not overlap (distinct CNV/CNV-subregion), butimpact the same gene (or regulatory locus) and are associated with an ORof greater than 6 (Genic (distinct CNV-subregions); OR >6). For example,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions do notoverlap, but impact the same gene (or regulatory locus), and areassociated with an OR of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 45, 50, or more. In some embodiments, CNVs/CNV-subregionscan be of interest if the CNVs/CNV-subregions do not overlap, but impactthe same gene (or regulatory locus), and are associated with an OR fromabout 6-100, 6-50, 6-40, 6-30, 6-20, 6-10, 6-9, 6-8, 6-7, 8-100, 8-50,8-40, 8-30, 8-20, 8-10, 10-100, 10-50, 10-40, 10-30, 10-20, 20-100,20-50, 20-40, 20-30, 30-100, 30-50, 30-40, 40-100, 40-50, 50-100, or5-7. The CNV-subregion/gene can be an exonic or intronic part of thegene, or both.

In some embodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions do not overlap a known gene (e.g., are non-genic orintergenic) and they are associated with an OR of at least 7 (Exon+ve,ASD >4, NVE <2, no Sanger filter applied). For example,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregion doesnot overlap a known gene (e.g., is non-genic or intergenic) and/ornon-overlapping, impact an exon, affect 5 or more ASD cases but only 0or 1 Normal subjects, no Sanger filter of CNVs is applied, and areassociated with an OR of at least 8, 9, 10, 11, 12, 14, 16, 18, 20, 25,30, 35, 40, 45, 50, or more. In some embodiments, CNVs/CNV-subregionscan be of interest if the CNVs/CNV-subregions are overlapping and/ornon-overlapping, impact an exon, affect 5 or more ASD cases but only 0or 1 Normal subjects, no filter of Sanger CNVs is applied, and areassociated with an OR from about 7-100, 7-50, 7-40, 7-30, 7-20, 20-100,20-50, 20-40, 20-30, 30-100, 30-50, 30-40, 40-100, 40-50, 50-100, or7-11.

In some embodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions are overlapping and/or non-overlapping, impact anexon, and they affect <5 ASD cases but only 0 or 1 Normal subjects, aSanger filter is applied, and there are no Sanger CNVs that overlap(Exon+ve, 5>ASD >1, Normals <2, Sanger filter −ve). This can enableidentification of rarer CNVs in cases with a neurodevelopmental disorderbut with the stringency of Sanger CNVs that are presumed to berelatively common in the general population. In some embodiments,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions areoverlapping and/or non-overlapping, impact an exon, and they affect 1ASD cases but only 0 or 1 Normal subjects, a Sanger filter is applied,there are no Sanger CNVs that overlap, and are associated with an ORgreater than 1, such as 1.47, or from 1-2.5. In some embodiments,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions areoverlapping and/or non-overlapping, impact an exon, and they affect 2ASD cases but only 0 or 1 Normal subjects, a Sanger filter is applied,there are no Sanger CNVs that overlap, and are associated with an ORgreater than 2.5, such as 2.95, or from 2.5-4. In some embodiments,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions areoverlapping and/or non-overlapping, impact an exon, and they affect 3ASD cases but only 0 or 1 Normal subjects, a Sanger filter is applied,there are no Sanger CNVs that overlap, and are associated with an ORgreater than 4, such as 4.44, or from 4-5.5. In some embodiments,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions areoverlapping and/or non-overlapping, impact an exon, and they affect 4ASD cases but only 0 or 1 Normal subjects, a Sanger filter is applied,there are no Sanger CNVs that overlap, and are associated with an ORgreater than 5.5, such as 5.92, or from 5.5-6.8

In some embodiments, a CNVs/CNV-subregions can be of interest if the ORassociated with the sum of ASD cases and the sum of NVE subjectsaffecting the same gene (including distinct CNVs/CNV-subregions) is atleast 6. For example, a CNV/CNV-subregion can be of interest if the ORassociated with the sum of ASD cases and the sum of NVE subjectsaffecting the same gene (including distinct CNVs/CNV-subregions) is atleast 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, or more.In some embodiments, a CNVs/CNV-subregions can be of interest if the ORassociated with the sum of ASD cases and the sum of NVE subjectsaffecting the same gene (including distinct CNVs/CNV-subregions) is fromabout 6-100, 6-50, 6-40, 6-30, 6-20, 6-10, 6-9, 6-8, 6-7, 8-100, 8-50.8-40, 8-30, 8-20, 8-10, 10-100, 10-50, 10-40, 10-30, 10-20, 20-100,20-50, 20-40, 20-30, 30-100, 30-50, 30-40, 40-100, 40-50, 50-100, or5-7.

In some embodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions impact an intron and they affect 5 or more ASD casesbut only 0 or 1 Normal subjects, no Sanger filter of CNVs is applied,and they are associated with an OR of at least 7 (Intron+ve, ASD >4,Normals <2, no Sanger filter applied). For example, CNVs/CNV-subregionscan be of interest if the CNVs/CNV-subregions impact an intron and theyaffect 5 or more ASD cases but only 0 or 1 Normal subjects, no Sangerfilter of CNVs is applied, and they are associated with an OR of atleast 8, 9, 10, 11, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, or more.In some embodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions impact an intron and they affect 5 or more ASD casesbut only 0 or 1 Normal subjects, no Sanger filter of CNVs is applied,and they are associated with an OR from about 7-100, 7-50, 7-40, 7-30,7-20, 20-100, 20-50, 20-40, 20-30, 30-100, 30-50, 30-40, 40-100, 40-50,50-100, or 7-11. CNVs/CNV-subregions impacting introns can be pathogenic(e.g., such variants can result in alternatively spliced mRNAs or lossof a microRNA binding site, which may deleteriously impact the resultingprotein's structure or expression level).

In some embodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions impact the MTRNR2L (also known as humanin) genefamily (MTRNR2L_family). While humanins may have neuroprotectiveproperties for Alzheimer's disease, it is not established inneurodevelopment disorders; however, recently links have beenestablished between the Alzheimer's gene APP and neurodevelopmentaldisorders such as autism (Westmark CJ. What's hAPPening at synapses? Therole of amyloid β-protein precursor and β-amyloid in neurologicaldisorders. Mol Psychiatry. 2012 Aug. 28). In some embodiments, a rareCNV of less than 0.2% frequency in a neurodevelopmental cohort can be ofinterest. For example, 1 ASD case may contain a CNV impacting a humaningene family member and this same CNV may not be found in a Normalsubject or in only 1 Normal subject such that the OR is 1.47. In anotherembodiment, the OR may be close to 1, such as 0.98, but with screeningof larger cohorts of Normal subjects and ASD cases (or otherneurodevelopmental cohort) for both CNVs and any other type of geneticvariant, such as SNVs via sequencing, it may be found that deleteriousmutations in a humanin gene occur at higher frequency inneurodevelopmental cases than in Normal subjects. In some embodimentsCNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions impactthe MTRNR2L1 gene and they affect 4 ASD cases but only 0 or 1 Normalsubjects and are associated with an OR greater than 5.5, such as greaterthan 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, ormore, or 5.92. In some embodiments CNVs/CNV-subregions can be ofinterest if the CNVs/CNV-subregions impact the MTRNR2L1 gene and theyaffect 4 ASD cases but only 0 or 1 Normal subjects and are associatedwith an OR from about 5.5-6. 8 In some embodiments CNVs/CNV-subregionscan be of interest if the CNVs/CNV-subregions impact the MTRNR2L4 geneand they affect 1 ASD case but only 0 or 1 Normal subjects and areassociated with an OR greater than 1, such greater than 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, or more, or1.47. In some embodiments CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions impact the MTRNR2L4 gene and they affect 1 ASD casebut only 0 or 1 Normal subjects and are associated with an OR from about1-2.5. In some embodiments CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions impact the MTRNR2L5 gene and they affect 2 ASD casesbut only 3 Normal subjects and are associated with an OR greater than0.5, such as greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16,18, 20, 25, 30, 35, 40, 45, 50, or more, or 0.98. In some embodimentsCNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions impactthe MTRNR2L5 gene and they affect 2 ASD cases but only 3 Normal subjectsand are associated with an OR from about 0.5-1. In some embodimentsCNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions impactthe MTRNR2L8 gene and they affect 1 ASD cases but only 0 or 1 Normalsubjects and are associated with an OR greater than 1, such as greaterthan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 25, 30, 35, 40,45, 50, or more, or 1.47. In some embodiments CNVs/CNV-subregions can beof interest if the CNVs/CNV-subregions impact the MTRNR2L8 gene and theyaffect 1 ASD cases but only 0 or 1 Normal subjects and are associatedwith an OR from about 1-2.5.

In some embodiments, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions occur within intergenic regions and are associatedwith an OR of greater than 30 (High OR intergenic (OR >30)). Forexample, CNVs/CNV-subregions can be of interest if theCNVs/CNV-subregions occur within intergenic regions and are associatedwith an OR of greater than 31, 32, 33, 34, 35, 40, 45, 50, 66, 60, 65,70, 75, 80, 85, 90, 95, 100 or more. In some embodiments,CNVs/CNV-subregions can be of interest if the CNVs/CNV-subregions impactoccur within intergenic regions and are associated with an OR from about30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80,40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90,60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100.

In some embodiments, a CNV/CNV-subregion can be of interest if theCNV/CNV-subregion overlaps a known gene, and is associated with an OR ofat least 10. In some embodiments, a CNV/CNV-subregion can be of interestif the CNV/CNV-subregion overlaps a known gene, is associated with an ORof at least 6, and if the OR associated with the sum of ASD cases andthe sum of NVE subjects affecting the same gene (including distinctCNV-subregions) is at least 6.

The data presented in Tables 1-4 was generated on the basis of acomparison of copy number variants (CNVs) identified in a NVE and an ASDcohort. CNV genome locations are provided using the Human March 2006(NCBI36/hg18) assembly. It can be appreciated by those skilled in theart that a CNV found in an affected individual may have one or moresubregions that are preferentially found in the affected cohort ascompared to the unaffected cohort and, similarly, other subregionswithin the CNV that are found at comparable frequencies, or notstatistically significant different frequencies, in the affected andunaffected cohorts. In a preferred embodiment, CNV detection andanalysis methods are employed that enable comparison of CNV subregionsto facilitate identification of genes (or regulatory loci) that arecausative or associated with the phenotype, condition, or disease beinginvestigated (or detected for diagnostic purposes)

Table 1 lists all CNVs (SEQ ID NOs: 1-883) of interest, obtained asdescribed in the text, with the exception that, for each entry, thechromosome (Chr) and original CNV start and stop positions are listed,along with original CNV size, type (loss or gain), ASD case ID, genesymbols (for the CNV-subregion, not the original CNV), Odds Ratio (OR)that is relevant to the CNV-subregion and, finally, the category ofinterest. The gene symbols refer to annotations for genes within theCNV-subregion, not the original CNV. In addition, the column ‘SEQ ID No’lists the SEQ IDs of the sequences being submitted. Note that for someCNVs that are identical between different individuals, the prioritynumbers (and SEQ IDs) are identical. In other words, the sequence for agiven CNV is only included once, if identical in different individuals.For example, 2 rows of Table 1 may refer to identical CNVs in 2 ASDcases.

Table 2 is identical to Table 1, with 4 exceptions. The CNV coordinateslisted refer to the actual CNV-subregions found to be unique orsignificantly different between the ASD and NVE cohorts, as opposed toTable 1, which lists the original CNVs. In addition, an extra columndetails whether genic CNV-subregions of interest overlap an exon or not(Exon Overlap, Y=yes, N=N). 2 extra columns detail the number of NVEsubjects (NVE) and the number of ASD cases (ASD) that harbor therelevant CNV-subregion.

Table 3 represents a non-redundant list for all genes listed in Table 2(namely, those relevant to CNV-subregions of interest), and includes theGene name (RefSeq Gene Symbol), Exon overlap (Y=yes, N=no), NCBI Gene ID(DNA Accession number), Gene Description (brief gene description), andRefSeq Summary (summary of gene function).

Table 4 represents a non-redundant list for all genes listed in Table 2(namely, those relevant to CNV-subregions of interest) and includesRefSeq Gene Symbol, Exon overlap (intronic, exonic or both, SEQ ID No(consecutive SEQ ID numbers from Table 1). SEQ ID NOs: 884-1690 refer tothe transcript sequences; RefSeq Accession Number (may be multipleentries per gene, hence Table 4 has more entries than Table 3);mRNA_Description (brief description of mRNA), and RefSeq Summmary(summary of gene function). For CNVs that encompass consecutive intronsand exons, there may be multiple features reported per CNV.

More than one RNA product (e.g., alternatively spliced mRNA transcriptsand non-coding RNAs) can be produced from a single gene. Table 4 listsall presently known transcript variants (and their RNA accessionnumbers) but new variants may be found when further studies arecompleted and that generation of these additional transcript variants(and ultimately polypeptide and/or regulatory RNA products) may also beimpacted by one or more CNVs or CNV subregions listed in Tables 1 and 2,respectively. The transcripts listed in Table 4 can be expressionproducts of the same gene biomarker. The gene biomarker can comprisegenomic DNA encoding the gene, including exons, introns, and/orregulatory binding regions (such as enhancers, promoters, silencers,and/or response elements). Point mutations, polymorphisms, singlenucleotide polymorphisms (SNPs), single nucleotide variations (SNVs),translocations, insertions, deletions, amplifications, inversions,microsatellites, interstitial deletions, CNVs, loss of heterozygosity,or any other aberrations which affect the structure or function of oneor more gene biomarkers and/or expression products thereof, can beassociated with a neurodevelopmental disorder as described herein.

Table 5 represents a key showing the relationship of the chromosomenumber in column 1 of Table 1 and Table 2 and the actual chromosomewhere the CNVs/CNV-subregions were detected.

In some embodiments, the CNVs from Table 1 only include the CNVs inTable 1 of U.S. Provisional Application No. 61/744,463.

In some embodiments, the CNV subregions from Table 2 only include theCNV subregions in Table 2 of U.S. Provisional Application No.61/744,463.

In some embodiments, the genes from Table 3 only include the genes inTable 3 of U.S. Provisional Application No. 61/744,463.

In some embodiments, the transcripts from Table 4 only include thetranscripts in Table 4 of U.S. Provisional Application No. 61/744,463.

Lengthy table referenced here US11618925-20230404-T00001 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US11618925-20230404-T00002 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US11618925-20230404-T00003 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US11618925-20230404-T00004 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US11618925-20230404-T00005 Please refer tothe end of the specification for access instructions.Computer-Implemented Aspects

As understood by those of ordinary skill in the art, the methods andinformation described herein (genetic variation association withdevelopmental disorders) can be implemented, in all or in part, ascomputer executable instructions on known computer readable media. Forexample, the methods described herein can be implemented in hardware.Alternatively, the method can be implemented in software stored in, forexample, one or more memories or other computer readable medium andimplemented on one or more processors. As is known, the processors canbe associated with one or more controllers, calculation units and/orother units of a computer system, or implanted in firmware as desired.If implemented in software, the routines can be stored in any computerreadable memory such as in RAM, ROM, flash memory, a magnetic disk, alaser disk, or other storage medium, as is also known. Likewise, thissoftware can be delivered to a computing device via any known deliverymethod including, for example, over a communication channel such as atelephone line, the Internet, a wireless connection, etc., or via atransportable medium, such as a computer readable disk, flash drive,etc.

More generally, and as understood by those of ordinary skill in the art,the various steps described above can be implemented as various blocks,operations, tools, modules and techniques which, in turn, can beimplemented in hardware, firmware, software, or any combination ofhardware, firmware, and/or software. When implemented in hardware, someor all of the blocks, operations, techniques, etc. can be implementedin, for example, a custom integrated circuit (IC), an applicationspecific integrated circuit (ASIC), a field programmable logic array(FPGA), a programmable logic array (PLA), etc.

Results from such genotyping can be stored in a data storage unit, suchas a data carrier, including computer databases, data storage disks, orby other convenient data storage means. In certain embodiments, thecomputer database is an object database, a relational database or apost-relational database. Data can be retrieved from the data storageunit using any convenient data query method.

When implemented in software, the software can be stored in any knowncomputer readable medium such as on a magnetic disk, an optical disk, orother storage medium, in a RAM or ROM or flash memory of a computer,processor, hard disk drive, optical disk drive, tape drive, etc.Likewise, the software can be delivered to a user or a computing systemvia any known delivery method including, for example, on a computerreadable disk or other transportable computer storage mechanism.

The steps of the claimed methods can be operational with numerous othergeneral purpose or special purpose computing system environments orconfigurations. Examples of well known computing systems, environments,and/or configurations that can be suitable for use with the methods orsystem of the claims include, but are not limited to, personalcomputers, server computers, hand-held or laptop devices, multiprocessorsystems, microprocessor-based systems, set top boxes, programmableconsumer electronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, and the like.

The steps of the claimed method and system can be described in thegeneral context of computer-executable instructions, such as programmodules, being executed by a computer. Generally, program modulesinclude routines, programs, objects, components, and/or data structuresthat perform particular tasks or implement particular abstract datatypes. The methods and apparatus can also be practiced in distributedcomputing environments where tasks are performed by remote processingdevices that are linked through a communications network. In bothintegrated and distributed computing environments, program modules canbe located in both local and remote computer storage media includingmemory storage devices. Numerous alternative embodiments could beimplemented, using either current technology or technology developedafter the filing date of this application, which would still fall withinthe scope of the claims defining the disclosure.

While the risk evaluation system and method, and other elements, havebeen described as preferably being implemented in software, they can beimplemented in hardware, firmware, etc., and can be implemented by anyother processor. Thus, the elements described herein can be implementedin a standard multi-purpose CPU or on specifically designed hardware orfirmware such as an application-specific integrated circuit (ASIC) orother hard-wired device as desired. When implemented in software, thesoftware routine can be stored in any computer readable memory such ason a magnetic disk, a laser disk, or other storage medium, in a RAM orROM of a computer or processor, in any database, etc. Likewise, thissoftware can be delivered to a user or a screening system via any knownor desired delivery method including, for example, on a computerreadable disk or other transportable computer storage mechanism or overa communication channel, for example, a telephone line, the internet, orwireless communication. Modifications and variations can be made in thetechniques and structures described and illustrated herein withoutdeparting from the spirit and scope of the present disclosure.

ASD Therapeutics

Research into a cure for Pervasive Developmental Disorders (PDD), suchas Autism Spectrum Disorder (ASD) or Pervasive DevelopmentalDisorders—Not Otherwise Specified (PDD-NOS), such as Asperger Syndrome,Rett Syndrome, Fragile X Syndrome, and/or Childhood DisintegrativeDisorder is ongoing. Ways to help minimize the symptoms of autism and tomaximize learning exist, including but not limited to, behavioraltherapy, educational and/or school-based options, and medicationoptions, although currently there are no medications that can cureautism spectrum disorders or all of the symptoms. The U.S. Food and DrugAdministration has not yet approved any medications specifically for thetreatment of autism, but in many cases medication can treat some of thesymptoms associated with autism. These treatments can include behaviormanagement therapy to help reinforce wanted behaviors and reduceunwanted behaviors, which is often based on Applied Behavior Analysis(ABA), use of speech-language therapists to help people with autismimprove their ability to communicate and interact with others, use ofoccupational therapists to help people find ways to adjust tasks tomatch their needs and abilities, and physical therapists designactivities and exercise to build motor control and improve posture andbalance, free appropriate public education from age 3 through highschool or age 21, integration of a team of people, including theparents, teachers, caregivers, school psychologists, and other childdevelopment specialists to work together to design an IndividualizedEducation Plan (IEP) to help guide the child's school experiences,selective serotonin reuptake inhibitors (SSRIs), tricyclics,psychoactive/anti-psychotics, stimulants, and anti-anxiety drugs areamong the medications that a health care provider might use to treatsymptoms of autism spectrum disorders.

A person skilled in the art will appreciate and understand that thegenetic variants described herein in general may not, by themselves,provide an absolute identification of individuals who can develop adevelopmental disorder or related conditions. The variants describedherein can indicate increased and/or decreased likelihood thatindividuals carrying the at-risk or protective variants of thedisclosure can develop symptoms associated with a developmentaldisorder. This information can be used to, for example, initiatepreventive measures at an early stage, perform regular physical and/ormental exams to monitor the progress and/or appearance of symptoms, orto schedule exams at a regular interval to identify early symptoms, soas to be able to apply treatment at an early stage. This is inparticular important since developmental disorders and related disordersare heterogeneous disorders with symptoms that can be individuallyvague. Screening criteria can comprise a number of symptoms to bepresent over a period of time; therefore, it is important to be able toestablish additional risk factors that can aid in the screening, orfacilitate the screening through in-depth phenotyping and/or morefrequent examination, or both. For example, individuals with earlysymptoms that typically are not individually associated with a clinicalscreening of a developmental disorder and carry an at-risk geneticvariation can benefit from early therapeutic treatment, or otherpreventive measure, or more rigorous supervision or more frequentexamination. Likewise, individuals that have a family history of thedisease, or are carriers of other risk factors associated with adevelopmental disorder can, in the context of additionally carrying atleast one at-risk genetic variation, benefit from early therapy or othertreatment.

Early symptoms of behavioral disorders such as a developmental disorderand related conditions may not be sufficient to fulfill standardizedscreening criteria. To fulfill those, a certain pattern of symptoms andbehavioral disturbance needs to manifest itself over a period of time.Sometimes, certain physical characteristics can also be present. Thismakes at-risk genetic variants valuable in a screening setting, inparticular high-risk variants. Determination of the presence of suchvariants warrants increased monitoring of the individual in question.Appearance of symptoms combined with the presence of such variantsfacilitates early screening, which makes early treatment possible.Genetic testing can thus be used to aid in the screening of disease inits early stages, before all criteria for formal screening criteria areall fulfilled. It is well established that early treatment is extremelyimportant for developmental disorders and related disorders, which lendsfurther support to the value of genetic testing for early diagnosis,prognosis, or theranosis of these disorders.

The present disclosure provides methods for identifying compounds oragents that can be used to treat a developmental disorder. Thus, thegenetic variations and associated polypeptides of the disclosure areuseful as targets for the identification and/or development oftherapeutic agents. In certain embodiments, such methods includeassaying the ability of an agent or compound to modulate the activityand/or expression of a nucleic acid that is associated with at least onegenetic variation described herein, encoded products of the genesequence, and any other molecules or polypeptides associated with thesegenes. This in turn can be used to identify agents or compounds thatinhibit, enhance, or alter the undesired activity, localization, bindingand/or expression of the encoded nucleic acid product, such as mRNA orpolypeptides. For example, in some embodiments, small molecule drugs canbe developed to target the aberrant polypeptide(s) or RNA(s) resultingfrom specific disease-causing mutation(s) within a gene, such asdescribed in: Peitz et al. (2009) RNA Biology 6(3):329-34; Van Goor etal. (2009) Proc. Natl. Acad. Sci. USA 106(44):18825-30; Van Goor et al.(2011) Proc. Natl. Acad. Sci. USA 108(46):18843-8; Ramsey et al. (2011)N. Engl. J. Med. 365(18):1663-72. The polypeptides associated with theCNVs listed in Table 1 are described in Table 4 as the accession number(accession) of mRNAs that would encode said polypeptides. Assays forperforming such experiments can be performed in cell-based systems or incell-free systems, as known to the skilled person. Cell-based systemsinclude cells naturally expressing the nucleic acids of interest, orrecombinant cells that have been genetically modified so as to express acertain desired nucleic acid molecule.

Variant gene expression in a subject can be assessed by expression of avariant-containing nucleic acid sequence or by altered expression of anormal/wild-type nucleic acid sequence due to variants affecting thelevel or pattern of expression of the normal transcripts, for example,variants in the regulatory or control region of the gene. Assays forgene expression include direct nucleic acid assays (mRNA), assays forexpressed polypeptide levels, or assays of collateral compounds involvedin a pathway, for example, a signal pathway. Furthermore, the expressionof genes that are up- or down-regulated in response to the signalpathway can also be assayed. One embodiment includes operably linking areporter gene, such as luciferase, to the regulatory region of one ormore gene of interest.

Modulators of gene expression can in some embodiments be identified whena cell is contacted with a candidate compound or agent, and theexpression of mRNA is determined. The expression level of mRNA in thepresence of the candidate compound or agent is compared to theexpression level in the absence of the compound or agent. Based on thiscomparison, candidate compounds or agents for treating a developmentaldisorder can be identified as those modulating the gene expression ofthe variant gene, or gene expression of one or more other genesoccurring within the same biological pathway or known, for example, tobe binding partners of the variant gene. When expression of mRNA or theencoded polypeptide is statistically significantly greater in thepresence of the candidate compound or agent than in its absence, thenthe candidate compound or agent is identified as a stimulator orup-regulator of expression of the nucleic acid. When nucleic acidexpression or polypeptide level is statistically significantly less inthe presence of the candidate compound or agent than in its absence,then the candidate compound can be identified as an inhibitor ordown-regulator of the nucleic acid expression. The disclosure furtherprovides methods of treatment using a compound identified through drug(compound and/or agent) screening as a gene modulator.

The genetic variations described herein can be used to identify noveltherapeutic targets for a developmental disorder. For example, genescontaining, or in linkage disequilibrium with, the genetic variations,or their products, as well as genes or their products that are directlyor indirectly regulated by or interact with these variant genes or theirproducts, can be targeted for the development of therapeutic agents totreat a developmental disorder, or prevent or delay onset of symptomsassociated with a developmental disorder. Therapeutic agents cancomprise one or more of, for example, small non-polypeptide andnon-nucleic acids, polypeptides, peptides, polypeptide fragments,nucleic acids (RNA, DNA, RNAJ, PNA (peptide nucleic acids), or theirderivatives or mimetics which can modulate the function and/or levels ofthe target genes or their gene products. In some embodiments, treatmentof ASD can comprise treatment of one of the genes, or gene productsderived thereof, such as mRNA or a polypeptide, with one or more of thetherapeutics disclosed herein. In some embodiments, treatment of ASD cancomprise treatment of 2 or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10or more of the genes, or gene products derived there from, with 2 or 3,or 4, or 5, or 6, or 7, or 8, or 9, or 10 or more of the therapeuticsdisclosed herein.

RNA Therapeutics

The nucleic acids and/or variants of the disclosure, or nucleic acidscomprising their complementary sequence, can be used as antisenseconstructs to control gene expression in cells, tissues or organs. Themethodology associated with antisense techniques is well known to theskilled artisan, and is described and reviewed in Antisense DrugTechnology: Principles, Strategies, and Applications, Crooke, MarcelDekker Inc., New York (2001) In general, antisense nucleic acids aredesigned to be complementary to a region of mRNA expressed by a gene, sothat the antisense molecule hybridizes to the mRNA, thus blockingtranslation of the mRNA into a polypeptide Several classes of antisenseoligonucleotide are known to those skilled in the art, includingcleavers and blockers. The former bind to target RNA sites, activateintracellular nucleases {e.g., Rnase H or Rnase L) that cleave thetarget RNA. Blockers bind to target RNA, inhibit polypeptide translationby steric hindrance of the ribosomes. Examples of blockers includenucleic acids, morpholino compounds, locked nucleic acids andmethylphosphonates (Thompson, Drug Discovery Today, 7:912-917 (2002))Antisense oligonucleotides are useful directly as therapeutic agents,and are also useful for determining and validating gene function, forexample, by gene knock-out or gene knock-down experiments. Antisensetechnology is further described in Lavery et al., Curr. Opin. DrugDiscov Devel 6 561-569 (2003), Stephens et al., Curr. Opin. Mol Ther.5.118-122 (2003), Kurreck, Eur. J. Biochem. 270.1628-44 (2003), Dias etal, Mol Cancer Ter. 1-347-55 (2002), Chen, Methods Mol Med. 75:621-636(2003), Wang et al., Curr Cancer Drug Targets 1.177-96 (2001), andBennett, Antisense Nucleic Acid Drug. Dev. 12 215-24 (2002)

The variants described herein can be used for the selection and designof antisense reagents that are specific for particular variants (e.g.,particular genetic variations, or polymorphic markers in LD withparticular genetic variations). Using information about the variantsdescribed herein, antisense oligonucleotides or other antisensemolecules that specifically target mRNA molecules that contain one ormore variants of the disclosure can be designed. In this manner,expression of mRNA molecules that contain one or more variant of thepresent disclosure (markers and/or haplotypes) can be inhibited orblocked In some embodiments, the antisense molecules are designed tospecifically bind a particular allelic form (i.e., one or severalvariants (alleles and/or haplotypes)) of the target nucleic acid,thereby inhibiting translation of a product originating from thisspecific allele or haplotype, but which do not bind other or alternatevariants at the specific polymorphic sites of the target nucleic acidmolecule.

As antisense molecules can be used to inactivate mRNA so as to inhibitgene expression, and thus polypeptide expression, the molecules can beused to treat a disease or disorder, such as a developmental disorder.The methodology can involve cleavage by means of ribozymes containingnucleotide sequences complementary to one or more regions in the mRNAthat attenuate the ability of the mRNA to be translated Such mRNAregions include, for example, polypeptide-coding regions, in particularpolypeptide-coding regions corresponding to catalytic activity,substrate and/or ligand binding sites, or other functional domains of apolypeptide.

The phenomenon of RNA interference (RNAi) has been actively studied forthe last decade, since its original discovery in C. elegans (Fire etal., Nature 391:806-11 (1998)), and in recent years its potential use intreatment of human disease has been actively pursued (reviewed in Kim &Rossi, Nature Rev, Genet. 8: 173-204 (2007)). RNA interference (RNAi),also called gene silencing, is based on using double-stranded RNAmolecules (dsRNA) to turn off specific genes. In the cell, cytoplasmicdouble-stranded RNA molecules (dsRNA) are processed by cellularcomplexes into small interfering RNA (siRNA). The siRNA guide thetargeting of a polypeptide-RNA complex to specific sites on a targetmRNA, leading to cleavage of the mRNA (Thompson, Drug Discovery Today,7:912-917 (2002)). The siRNA molecules are typically about 20, 21, 22 or23 nucleotides in length. Thus, one aspect of the disclosure relates toisolated nucleic acid sequences, and the use of those molecules for RNAinterference, for example, as small interfering RNA molecules (siRNA).In some embodiments, the isolated nucleic acid sequences can be 18-26nucleotides in length, preferably 19-25 nucleotides in length, morepreferably 20-24 nucleotides in length, and more preferably 21, 22 or 23nucleotides in length.

Another pathway for RNAi-mediated gene silencing originates inendogenously encoded primary microRNA (pn-miRNA) transcripts, which areprocessed in the cell to generate precursor miRNA (pre-miRNA). ThesemiRNA molecules are exported from the nucleus to the cytoplasm, wherethey undergo processing to generate mature miRNA molecules (miRNA),which direct translational inhibition by recognizing target sites in the3′ untranslated regions of mRNAs, and subsequent mRNA degradation byprocessing P-bodies (reviewed in Kim & Rossi, Nature Rev. Genet. 8:173-204 (2007)).

Clinical applications of RNAi include the incorporation of syntheticsiRNA duplexes, which preferably are approximately 20-23 nucleotides insize, and preferably have 3′ overlaps of 2 nucleotides. Knockdown ofgene expression is established by sequence-specific design for thetarget mRNA. Several commercial sites for optimal design and synthesisof such molecules are known to those skilled in the art.

Other applications provide longer siRNA molecules (typically 25-30nucleotides in length, preferably about 27 nucleotides), as well assmall hairpin RNAs (shRNAs; typically about 29 nucleotides in length).The latter are naturally expressed, as described in Amarzguioui et al.(FEBS Lett. 579:5974-81 (2005)). Chemically synthetic siRNAs and shRNAsare substrates for in vivo processing, and in some cases provide morepotent gene-silencing than shorter designs (Kim et al., NatureBiotechnol. 23:222-226 (2005); Siola et al., Nature Biotechnol.23:227-231 (2005)). In general siRNAs provide for transient silencing ofgene expression, because their intracellular concentration is diluted bysubsequent cell divisions. By contrast, expressed shRNAs mediatelong-term, stable knockdown of target transcripts, for as long astranscription of the shRNA takes place (Marques et al., NatureBiotechnol. 23.559-565 (2006), Brummelkamp et al., Science 296. 550-553(2002)).

Since RNAi molecules, including siRNA, miRNA and shRNA, act in asequence-dependent manner, variants described herein can be used todesign RNAi reagents that recognize specific nucleic acids comprisingspecific genetic variations, alleles and/or haplotypes, while notrecognizing nucleic acid sequences not comprising the genetic variation,or comprising other alleles or haplotypes. These RNAi reagents can thusrecognize and destroy the target nucleic acid sequences. As withantisense reagents, RNAi reagents can be useful as therapeutic agents(i.e., for turning off disease-associated genes or disease-associatedgene variants), but can also be useful for characterizing and validatinggene function (e.g., by gene knock-out or gene knock-down experiments).

Delivery of RNAi can be performed by a range of methodologies known tothose skilled in the art. Methods utilizing non-viral delivery includecholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chainantibody fragment (Fab), aptamers and nanoparticles Viral deliverymethods include use of lentivirus, adenovirus and adeno-associated virusThe siRNA molecules are in some embodiments chemically modified toincrease their stability. This can include modifications at the 2′position of the ribose, including 2′-O-methylpunnes and2′-fluoropyrimidmes, which provide resistance to RNase activity. Otherchemical modifications are possible and known to those skilled in theart.

The following references provide a further summary of RNAi, andpossibilities for targeting specific genes using RNAi: Kim & Rossi, Nat.Rev. Genet. 8: 173-184 (2007), Chen & Rajewsky, Nat. Rev. Genet. 8:93-103 (2007), Reynolds, et al., Nat. Biotechnol 22 326-330 (2004), Chiet al., Proc. Natl. Acad. Sa. USA 100-6343-6346 (2003), Vickers et al.,J Biol Chem. 278:7108-7118 (2003), Agami, Curr Opin. Chem. Biol.6:829-834 (2002), Lavery, et al., Curr. Opin. Drug Discov. Devel.6:561-569 (2003), Shi, Trends Genet. 19:9-12 (2003), Shuey et al., DrugDiscov. Today 7 1040-46 (2002), McManus et al., Nat. Rev. Genet.3.737-747 (2002), Xia et al., Nat. Biotechnol. 20.1006-10 (2002),Plasterk et al., Curr. Opin Genet. Dev. 10 562-7 (2000), Bosher et al.,Nat. Cell Biol. 2:E31-6 (2000), and Hunter, Curr. Biol. 9:R440-442(1999).

A genetic defect leading to increased predisposition or risk fordevelopment of a disease, including a developmental disorder, or adefect causing the disease, can be corrected permanently byadministering to a subject carrying the defect a nucleic acid fragmentthat incorporates a repair sequence that supplies the normal/wild-typenucleotide(s) at the site of the genetic defect. Such site-specificrepair sequence can encompass an RNA/DNA oligonucleotide that operatesto promote endogenous repair of a subject's genomic DNA. Theadministration of the repair sequence can be performed by an appropriatevehicle, such as a complex with polyethelamine, encapsulated in anionicliposomes, a viral vector such as an adenovirus vector, or otherpharmaceutical compositions suitable for promoting intracellular uptakeof the administered nucleic acid The genetic defect can then beovercome, since the chimeric oligonucleotides induce the incorporationof the normal sequence into the genome of the subject, leading toexpression of the normal/wild-type gene product. The replacement ispropagated, thus rendering a permanent repair and alleviation of thesymptoms associated with the disease or condition.

Double stranded oligonucleotides are formed by the assembly of twodistinct oligonucleotide sequences where the oligonucleotide sequence ofone strand is complementary to the oligonucleotide sequence of thesecond strand; such double stranded oligonucleotides are generallyassembled from two separate oligonucleotides (e.g., siRNA), or from asingle molecule that folds on itself to form a double stranded structure(e.g., shRNA or short hairpin RNA). These double strandedoligonucleotides known in the art all have a common feature in that eachstrand of the duplex has a distinct nucleotide sequence, wherein onlyone nucleotide sequence region (guide sequence or the antisensesequence) has complementarity to a target nucleic acid sequence and theother strand (sense sequence) comprises nucleotide sequence that ishomologous to the target nucleic acid sequence.

Double stranded RNA induced gene silencing can occur on at least threedifferent levels: (i) transcription inactivation, which refers to RNAguided DNA or histone methylation; (ii) siRNA induced mRNA degradation;and (iii) mRNA induced transcriptional attenuation. It is generallyconsidered that the major mechanism of RNA induced silencing (RNAinterference, or RNAi) in mammalian cells is mRNA degradation. RNAinterference (RNAi) is a mechanism that inhibits gene expression at thestage of translation or by hindering the transcription of specificgenes. Specific RNAi pathway polypeptides are guided by the dsRNA to thetargeted messenger RNA (mRNA), where they “cleave” the target, breakingit down into smaller portions that can no longer be translated into apolypeptide. Initial attempts to use RNAi in mammalian cells focused onthe use of long strands of dsRNA. However, these attempts to induce RNAimet with limited success, due in part to the induction of the interferonresponse, which results in a general, as opposed to a target-specific,inhibition of polypeptide synthesis. Thus, long dsRNA is not a viableoption for RNAi in mammalian systems. Another outcome is epigeneticchanges to a gene—histone modification and DNA methylation—affecting thedegree the gene is transcribed.

More recently it has been shown that when short (18-30 bp) RNA duplexesare introduced into mammalian cells in culture, sequence-specificinhibition of target mRNA can be realized without inducing an interferonresponse. Certain of these short dsRNAs, referred to as small inhibitoryRNAs (“siRNAs”), can act catalytically at sub-molar concentrations tocleave greater than 95% of the target mRNA in the cell. A description ofthe mechanisms for siRNA activity, as well as some of its applicationsare described in Provost et al., Ribonuclease Activity and RNA Bindingof Recombinant Human Dicer, E.M.B.O. J., 2002 Nov. 1; 21(21): 5864-5874;Tabara et al., The dsRNA Binding Protein RDE-4 Interacts with RDE-1,DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 2002,June 28; 109(7):861-71; Ketting et al., Dicer Functions in RNAInterference and in Synthesis of Small RNA Involved in DevelopmentalTiming in C. elegans; Martinez et al., Single-Stranded Antisense siRNAsGuide Target RNA Cleavage in RNAi, Cell 2002, September. 6; 110(5):563;Hutvagner & Zamore, A microRNA in a multiple-turnover RNAi enzymecomplex, Science 2002, 297:2056.

From a mechanistic perspective, introduction of long double stranded RNAinto plants and invertebrate cells is broken down into siRNA by a TypeIII endonuclease known as Dicer. Sharp, RNA interference—2001, GenesDev. 2001, 15:485. Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs. Bernstein, Caudy, Hammond, & Hannon, Role for abidentate ribonuclease in the initiation step of RNA interference,Nature 2001, 409:363. The siRNAs are then incorporated into anRNA-induced silencing complex (RISC) where one or more helicases unwindthe siRNA duplex, enabling the complementary antisense strand to guidetarget recognition (Nykanen, Haley, & Zamore, ATP requirements and smallinterfering RNA structure in the RNA interference pathway, Cell 2001,107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleaves the target to induce silencing.Elbashir, Lendeckel, & Tuschl, RNA interference is mediated by 21- and22-nucleotide RNAs, Genes Dev 2001, 15:188, FIG. 1 .

Generally, the antisense sequence is retained in the active RISC complexand guides the RISC to the target nucleotide sequence by means ofcomplementary base-pairing of the antisense sequence with the targetsequence for mediating sequence-specific RNA interference. It is knownin the art that in some cell culture systems, certain types ofunmodified siRNAs can exhibit “off target” effects. It is hypothesizedthat this off-target effect involves the participation of the sensesequence instead of the antisense sequence of the siRNA in the RISCcomplex (see for example, Schwarz et al., 2003, Cell, 115, 199-208). Inthis instance the sense sequence is believed to direct the RISC complexto a sequence (off-target sequence) that is distinct from the intendedtarget sequence, resulting in the inhibition of the off-target sequence.In these double stranded nucleic acid sequences, each strand iscomplementary to a distinct target nucleic acid sequence. However, theoff-targets that are affected by these dsRNAs are not entirelypredictable and are non-specific.

The term “siRNA” refers to small inhibitory RNA duplexes that induce theRNA interference (RNAi) pathway. These molecules can vary in length(generally between 18-30 basepairs) and contain varying degrees ofcomplementarity to their target mRNA in the antisense strand. Some, butnot all, siRNA have unpaired overhanging bases on the 5′ or 3′ end ofthe sense strand and/or the antisense strand. The term “siRNA” includesduplexes of two separate strands, as well as single strands that canform hairpin structures comprising a duplex region. Small interferingRNA (siRNA), sometimes known as short interfering RNA or silencing RNA,are a class of 20-25 nucleotide-long double-stranded RNA molecules thatplay a variety of roles in biology.

While the two RNA strands do not need to be completely complementary,the strands should be sufficiently complementary to hybridize to form aduplex structure. In some instances, the complementary RNA strand can beless than 30 nucleotides, preferably less than 25 nucleotides in length,more preferably 19 to 24 nucleotides in length, more preferably 20-23nucleotides in length, and even more preferably 22 nucleotides inlength. The dsRNA of the present disclosure can further comprise atleast one single-stranded nucleotide overhang. The dsRNA of the presentdisclosure can further comprise a substituted or chemically modifiednucleotide. As discussed in detail below, the dsRNA can be synthesizedby standard methods known in the art.

siRNA can be divided into five (5) groups including non-functional,semi-functional, functional, highly functional, and hyper-functionalbased on the level or degree of silencing that they induce in culturedcell lines. As used herein, these definitions are based on a set ofconditions where the siRNA is transfected into the cell line at aconcentration of 100 nM and the level of silencing is tested at a timeof roughly 24 hours after transfection, and not exceeding 72 hours aftertransfection. In this context, “non-functional siRNA” are defined asthose siRNA that induce less than 50% (<50%) target silencing.“Semi-functional siRNA” induce 50-79% target silencing. “FunctionalsiRNA” are molecules that induce 80-95% gene silencing.“Highly-functional siRNA” are molecules that induce greater than 95%gene silencing. “Hyperfunctional siRNA” are a special class ofmolecules. For purposes of this document, hyperfunctional siRNA aredefined as those molecules that: (1) induce greater than 95% silencingof a specific target when they are transfected at subnanomolarconcentrations (i.e., less than one nanomolar); and/or (2) inducefunctional (or better) levels of silencing for greater than 96 hours.These relative functionalities (though not intended to be absolutes) canbe used to compare siRNAs to a particular target for applications suchas functional genomics, target identification and therapeutics.

microRNAs (miRNA) are single-stranded RNA molecules of about 21-23nucleotides in length, which regulate gene expression. miRNAs areencoded by genes that are transcribed from DNA but not translated into apolypeptide (non-coding RNA); instead they are processed from primarytranscripts known as pri-miRNA to short stem-loop structures calledpre-miRNA and finally to functional miRNA. Mature miRNA molecules arepartially complementary to one or more messenger RNA (mRNA) molecules,and their main function is to downregulate gene expression.

Antibody-Based Therapeutics

The present disclosure embodies agents that modulate a peptide sequenceor RNA expressed from a gene associated with a developmental disorder.The term “biomarker”, as used herein, can comprise a genetic variationof the present disclosure or a gene product, for example, RNA andpolypeptides, of any one of the genes listed in Tables 1-4. Suchmodulating agents include, but are not limited to, polypeptides,peptidomimetics, peptoids, or any other forms of a molecule, which bindto, and alter the signaling or function associated with the adevelopmental disorder associated biomarker, have an inhibitory orstimulatory effect on the developmental disorder associated biomarkers,or have a stimulatory or inhibitory effect on the expression or activityof the a developmental disorder associated biomarkers' ligands, forexample, polyclonal antibodies and/or monoclonal antibodies thatspecifically bind one form of the gene product but not to the other formof the gene product are also provided, or which bind a portion of eitherthe variant or the reference gene product that contains the polymorphicsite or sites.

In some embodiments, the present disclosure provides antibody-basedagents targeting a developmental disorder associated biomarkers. Theantibody-based agents in any suitable form of an antibody e.g.,monoclonal, polyclonal, or synthetic, can be utilized in the therapeuticmethods disclosed herein. The antibody-based agents include anytarget-binding fragment of an antibody and also peptibodies, which areengineered therapeutic molecules that can bind to human drug targets andcontain peptides linked to the constant domains of antibodies. In someembodiments, the antibodies used for targeting a developmental disorderassociated biomarkers are humanized antibodies. Methods for humanizingantibodies are well known in the art. In some embodiments, thetherapeutic antibodies comprise an antibody generated against adevelopmental disorder associated biomarkers described in the presentdisclosure, wherein the antibodies are conjugated to another agent oragents, for example, a cytotoxic agent or agents.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain antigen-binding sites that specifically bind anantigen. A molecule that specifically binds to a polypeptide of thedisclosure is a molecule that binds to that polypeptide or a fragmentthereof, but does not substantially bind other molecules in a nucleicacid sample, which naturally contains the polypeptide. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin. The disclosure providespolyclonal and monoclonal antibodies that bind to a polypeptide of thedisclosure. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of a polypeptide ofthe disclosure. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular polypeptide of thedisclosure with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a desired immunogen, e.g., polypeptide of thedisclosure or a fragment thereof. The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody molecules directed against thepolypeptide can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybndoma technique originally described by Kohler and Milstein, Nature256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al.,Immunol. Today 4: 72 (1983)), the EBV-hybndoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985) Inc., pp.77-96) or trioma techniques. The technology for producing hybndomas iswell known (see generally Current Protocols in Immunology (1994) Coliganet al., (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, animmortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with an immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds a polypeptide of the disclosure.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to a polypeptide of the disclosure (see, e.g.,Current Protocols in Immunology, supra; Galfre et al., Nature 266:55052(1977); R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); andLerner, Yale J. Biol. Med. 54:387-402 (1981)). Moreover, the ordinarilyskilled worker can appreciate that there are many variations of suchmethods that also would be useful. Alternative to preparing monoclonalantibody-secreting hybridomas, a monoclonal antibody to a polypeptide ofthe disclosure can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the polypeptide to thereby isolate immunoglobulin librarymembers that bind the polypeptide. Kits for generating and screeningphage display libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAPa Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO92/18619, WO 91/17271, WO 92/20791, WO 92/15679; WO 93/01288, WO92/01047, WO 92/09690, and WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybndomas 3:81-85 (1992);Huse et al., Science 246: 1275-1281 (1989); and Griffiths et al., EMBOJ. 12:725-734 (1993).

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the disclosure. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

In general, antibodies of the disclosure (e.g., a monoclonal antibody)can be used to isolate a polypeptide of the disclosure by standardtechniques, such as affinity chromatography or immunoprecipitation. Apolypeptide-specific antibody can facilitate the purification of naturalpolypeptide from cells and of recombinants produced polypeptideexpressed in host cells Moreover, an antibody specific for a polypeptideof the disclosure can be used to detect the polypeptide (e.g., in acellular lysate, cell supernatant, or tissue sample) in order toevaluate the abundance and pattern of expression of the polypeptide.Antibodies can be used diagnostically, prognostically, ortheranostically to monitor polypeptide levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. The antibody can be coupled to adetectable substance to facilitate its detection. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotnazinylamine fluorescein, dansylchloride or phycoerythnn; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H. Antibodies can also be useful inpharmacogenomic analysis. In such embodiments, antibodies againstvariant polypeptides encoded by nucleic acids according to thedisclosure, such as variant polypeptides that are encoded by nucleicacids that contain at least one genetic variation of the disclosure, canbe used to identify individuals that can benefit from modified treatmentmodalities.

Antibodies can furthermore be useful for assessing expression of variantpolypeptides in disease states, such as in active stages of a disease,or in an individual with a predisposition to a disease related to thefunction of the polypeptide, in particular a developmental disorder.Antibodies specific for a variant polypeptide of the present disclosurethat is encoded by a nucleic acid that comprises at least onepolymorphic marker or haplotype as described herein can be used toscreen for the presence of the variant polypeptide, for example, toscreen for a predisposition to a developmental disorder as indicated bythe presence of the variant polypeptide.

Antibodies can be used in other methods. Thus, antibodies are useful asscreening tools for evaluating polypeptides, such as variantpolypeptides of the disclosure, in conjunction with analysis byelectrophoretic mobility, isoelectric point, tryptic or other proteasedigest, or for use in other physical assays known to those skilled inthe art. Antibodies can also be used in tissue typing. In one suchembodiment, a specific variant polypeptide has been correlated withexpression in a specific tissue type, and antibodies specific for thevariant polypeptide can then be used to identify the specific tissuetype.

Subcellular localization of polypeptides, including variantpolypeptides, can also be determined using antibodies, and can beapplied to assess aberrant subcellular localization of the polypeptidein cells in various tissues. Such use can be applied in genetic testing,but also in monitoring a particular treatment modality. In the casewhere treatment is aimed at correcting the expression level or presenceof the variant polypeptide or aberrant tissue distribution ordevelopmental expression of the variant polypeptide, antibodies specificfor the variant polypeptide or fragments thereof can be used to monitortherapeutic efficacy.

Antibodies are further useful for inhibiting variant polypeptidefunction, for example, by blocking the binding of a variant polypeptideto a binding molecule or partner. Such uses can also be applied in atherapeutic context in which treatment involves inhibiting a variantpolypeptide's function. An antibody can be for example, be used to blockor competitively inhibit binding, thereby modulating (i.e., agonizing orantagonizing) the activity of the polypeptide. Antibodies can beprepared against specific polypeptide fragments containing sites forspecific function or against an intact polypeptide that is associatedwith a cell or cell membrane.

The present disclosure also embodies the use of any pharmacologic agentthat can be conjugated to an antibody or an antibody binding fragment,and delivered in active form. Examples of such agents includecytotoxins, radioisotopes, hormones such as a steroid, anti-metabolitessuch as cytosines, and chemotherapeutic agents. Other embodiments caninclude agents such as a coagulant, a cytokine, growth factor, bacterialendotoxin or a moiety of bacterial endotoxin. The targetingantibody-based agent directs the toxin to, and thereby selectivelymodulates the cell expressing the targeted surface receptor. In someembodiments, therapeutic antibodies employ cross-linkers that providehigh in vivo stability (Thorpe et al., Cancer Res., 48:6396, 1988). Inany event, it is proposed that agents such as these can, if desired, besuccessfully conjugated to antibodies or antibody binding fragments, ina manner that can allow their targeting, internalization, release orpresentation at the site of the targeted cells expressing the ASDassociated biomarkers using known conjugation technology. Foradministration in vivo, for example, an antibody can be linked with anadditional therapeutic payload, such as radionuclide, an enzyme, animmunogenic epitope, or a cytotoxic agent, including bacterial toxins(diphtheria or plant toxins, such as ricin). The in vivo half-life of anantibody or a fragment thereof can be increased by pegylation throughconjugation to polyethylene glycol.

Gene Therapy

In some embodiments, gene therapy can be used as a therapeutic tomodulate a peptide sequence or RNA expressed from a gene associated witha developmental disorder. Gene therapy involves the use of DNA as apharmaceutical agent to treat disease. DNA can be used to supplement oralter genes within an individual's cells as a therapy to treat disease.Gene therapy can be used to alter the signaling or function associatedwith the a developmental disorder associated biomarker, have aninhibitory or stimulatory effect on the developmental disorderassociated biomarkers, or have a stimulatory or inhibitory effect on theexpression or activity of the a developmental disorder associatedbiomarkers' ligands. In one embodiment, gene therapy involves using DNAthat encodes a functional, therapeutic gene in order to replace amutated gene. Other forms involve directly correcting a mutation, orusing DNA that encodes a therapeutic polypeptide drug (rather than anatural human gene) to provide treatment. DNA that encodes a therapeuticpolypeptide can be packaged within a vector, which can used to introducethe DNA inside cells within the body. Once inside, the DNA becomesexpressed by the cell machinery, resulting in the production of thetherapeutic, which in turn can treat the subject's disease.

Gene therapy agents and other agents for testing therapeutics caninclude plasmids, viral vectors, artificial chromosomes and the likecontaining therapeutic genes or polynucleotides encoding therapeuticproducts, including coding sequences for small interfering RNA (siRNA),ribozymes and antisense RNA, which in certain further embodiments cancomprise an operably linked promoter such as a constitutive promoter ora regulatable promoter, such as an inducible promoter (e.g., IPTGinducible), a tightly regulated promoter (e.g., a promoter that permitslittle or no detectable transcription in the absence of its cognateinducer or derepressor) or a tissue-specific promoter. Methodologies forpreparing, testing and using these and related agents are known in theart. See, e.g., Ausubel (Ed.), Current Protocols in Molecular Biology(2007 John Wiley & Sons, NY); Rosenzweig and Nabel (Eds), CurrentProtocols in Human Genetics (esp. Ch. 13 therein, “Delivery Systems forGene Therapy”, 2008 John Wiley & Sons, NY); Abell, Advances in AminoAcid Mimetics and Peptidomimetics, 1997 Elsevier, NY. In anotherembodiment, gene therapy agents may encompass zinc finger nuclease (ZFN)or transcription activator-like effector nuclease (TALEN) strategies,see for example: Urnov et al. (2010), Nature Reviews Genetics11(9):636-46; Yusa et al. (2011), Nature 478(7369):391-4; Bedell et al.(2012), Nature ePub September 23, PubMed ID 23000899.

As a non-limiting example, one such embodiment contemplates introductionof a gene therapy agent for treating ASD (e.g., an engineeredtherapeutic virus, a therapeutic agent-carrying nanoparticle, etc.) toone or more injection sites in a subject, without the need for imaging,surgery, or histology on biopsy specimens. Of course, periodicmonitoring of the circulation for leaked therapeutic agent and/orsubsequent analysis of a biopsy specimen, e.g., to assess the effects ofthe agent on the target tissue, can also be considered. A gene therapyincludes a therapeutic polynucleotide administered before, after, or atthe same time as any other therapy described herein. In someembodiments, therapeutic genes may include an antisense version of abiomarker disclosed herein, a sequence of a biomarker described herein,or an inhibitor of a biomarker disclosed herein.

Methods of Treatment

Some embodiments of the present disclosure relates to methods of usingpharmaceutical compositions and kits comprising agents that can inhibita developmental disorder associated biomarker or a developmentaldisorder associated biomarkers to inhibit or decrease a developmentaldisorder progression. Another embodiment of the present disclosureprovides methods, pharmaceutical compositions, and kits for thetreatment of animal subjects. The term “animal subject” as used hereinincludes humans as well as other mammals. The term “treating” as usedherein includes achieving a therapeutic benefit and/or a prophylacticbenefit. By therapeutic benefit is meant eradication or amelioration ofthe underlying viral infection. Also, a therapeutic benefit is achievedwith the eradication or amelioration of one or more of the physiologicalsymptoms associated a developmental disorder such that an improvement isobserved in the animal subject, notwithstanding the fact that the animalsubject can still be afflicted with a developmental disorder.

For embodiments where a prophylactic benefit is desired, apharmaceutical composition of the disclosure can be administered to asubject at risk of developing a developmental disorder, or to a subjectreporting one or more of the physiological symptoms of a developmentaldisorder, even though a screening of the condition cannot have beenmade. Administration can prevent a developmental disorder fromdeveloping, or it can reduce, lessen, shorten and/or otherwiseameliorate the progression of a developmental disorder, or symptoms thatdevelop. The pharmaceutical composition can modulate or target adevelopmental disorder associated biomarker. Wherein, the term modulateincludes inhibition of a developmental disorder associated biomarkers oralternatively activation of a developmental disorder associatedbiomarkers.

Reducing the activity of a developmental disorder associated biomarkersis also referred to as “inhibiting” the a developmental disorderassociated biomarkers. The term “inhibits” and its grammaticalconjugations, such as “inhibitory,” do not require complete inhibition,but refer to a reduction in a developmental disorder associatedbiomarkers' activities. In some embodiments such reduction is by atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 75%, at least 90%, and can be by at least 95% of theactivity of the enzyme or other biologically important molecular processin the absence of the inhibitory effect, e.g., in the absence of aninhibitor. Conversely, the phrase “does not inhibit” and its grammaticalconjugations refer to situations where there is less than 20%, less than10%, and can be less than 5%, of reduction in enzyme or otherbiologically important molecular activity in the presence of the agent.Further the phrase “does not substantially inhibit” and its grammaticalconjugations refer to situations where there is less than 30%, less than20%, and in some embodiments less than 10% of reduction in enzyme orother biologically important molecular activity in the presence of theagent.

Increasing the activity a developmental disorderand/or function ofpolypeptides and/or nucleic acids found to be associated with one ormore developmentaldisorders, is also referred to as “activating” thepolypeptides and/or nucleic acids. The term “activated” and itsgrammatical conjugations, such as “activating,” do not require completeactivation, but refer to an increase in a developmental disorderassociated biomarkers' activities. In some embodiments such increase isby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, and can be by at least 95% of the activity ofthe enzyme or other biologically important molecular process in theabsence of the activation effect, e.g., in the absence of an activator.Conversely, the phrase “does not activate” and its grammaticalconjugations refer to situations where there can be less than 20%, lessthan 10%, and less than 5%, of an increase in enzyme or otherbiologically important molecular activity in the presence of the agent.Further the phrase “does not substantially activate” and its grammaticalconjugations refer to situations where there is less than 30%, less than20%, and in some embodiments less than 10% of an increase in enzyme orother biologically important molecular activity in the presence of theagent.

The ability to reduce enzyme activity is a measure of the potency or theactivity of an agent, or combination of agents, towards or against theenzyme or other biologically important molecular process. Potency can bemeasured by cell free, whole cell and/or in vivo assays in terms ofIC50, Ki and/or ED50 values. An IC50 value represents the concentrationof an agent required to inhibit enzyme activity by half (50%) under agiven set of conditions. A Ki value represents the equilibrium affinityconstant for the binding of an inhibiting agent to the enzyme or otherrelevant biomolecule. An ED50 value represents the dose of an agentrequired to affect a half-maximal response in a biological assay.Further details of these measures will be appreciated by those ofordinary skill in the art, and can be found in standard texts onbiochemistry, enzymology, and the like.

The present disclosure also includes kits that can be used to treatdevelopmental disorders. These kits comprise an agent or combination ofagents that inhibits a developmental disorder associated biomarker or adevelopmental disorder associated biomarkers and in some embodimentsinstructions teaching the use of the kit according to the variousmethods and approaches described herein. Such kits can also includeinformation, such as scientific literature references, package insertmaterials, clinical trial results, and/or summaries of these and thelike, which indicate or establish the activities and/or advantages ofthe agent. Such information can be based on the results of variousstudies, for example, studies using experimental animals involving invivo models and studies based on human clinical trials. Kits describedherein can be provided, marketed and/or promoted to health providers,including physicians, nurses, pharmacists, formulary officials, and thelike.

In some aspects a host cell can be used for testing or administeringtherapeutics. In some embodiments, a host cell can comprise a nucleicacid comprising expression control sequences operably-linked to a codingregion. The host cell can be natural or non-natural. The non-naturalhost used in aspects of the method can be any cell capable of expressinga nucleic acid of the disclosure including, bacterial cells, fungalcells, insect cells, mammalian cells and plant cells. In some aspectsthe natural host is a mammalian tissue cell and the non-natural host isa different mammalian tissue cell. Other aspects of the method include anatural host that is a first cell normally residing in a first mammalianspecies and the non-natural host is a second cell normally residing in asecond mammalian species. In another alternative aspect, the method usesa first cell and the second cell that are from the same tissue type. Inthose aspects of the method where the coding region encodes a mammalianpolypeptide, the mammalian polypeptide may be a hormone. In otheraspects the coding region may encode a neuropeptide, an antibody, anantimetabolite, or a polypeptide or nucleotide therapeutic.

Expression control sequences can be those nucleotide sequences, both 5′and 3′ to a coding region, that are required for the transcription andtranslation of the coding region in a host organism. Regulatorysequences include a promoter, ribosome binding site, optional inducibleelements and sequence elements required for efficient 3′ processing,including polyadenylation. When the structural gene has been isolatedfrom genomic DNA, the regulatory sequences also include those intronicsequences required for splicing of the introns as part of mRNA formationin the target host.

Formulations, Routes of Administration, and Effective Doses

Yet another aspect of the present disclosure relates to formulations,routes of administration and effective doses for pharmaceuticalcompositions comprising an agent or combination of agents of the instantdisclosure. Such pharmaceutical compositions can be used to treat adevelopmental disorder progression and a developmental disorderassociated symptoms as described above.

Compounds of the disclosure can be administered as pharmaceuticalformulations including those suitable for oral (including buccal andsub-lingual), rectal, nasal, topical, transdermal patch, pulmonary,vaginal, suppository, or parenteral (including intramuscular,intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneousand intravenous) administration or in a form suitable for administrationby aerosolization, inhalation or insufflation. General information ondrug delivery systems can be found in Ansel et al., PharmaceuticalDosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins,Baltimore Md. (1999).

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, polypeptides, amino acids, antioxidants, bacteriostats,chelating agents, suspending agents, thickening agents and/orpreservatives), water, oils including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like, saline solutions, aqueous dextrose andglycerol solutions, flavoring agents, coloring agents, detackifiers andother acceptable additives, adjuvants, or binders, otherpharmaceutically acceptable auxiliary substances to approximatephysiological conditions, such as pH buffering agents, tonicityadjusting agents, emulsifying agents, wetting agents and the like.Examples of excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. In some embodiments, thepharmaceutical preparation is substantially free of preservatives. Inother embodiments, the pharmaceutical preparation can contain at leastone preservative. General methodology on pharmaceutical dosage forms isfound in Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems (Lippencott, Williams, & Wilkins, Baltimore Md. (1999)). It canbe recognized that, while any suitable carrier known to those ofordinary skill in the art can be employed to administer the compositionsof this disclosure, the type of carrier can vary depending on the modeof administration.

Compounds can also be encapsulated within liposomes using well-knowntechnology. Biodegradable microspheres can also be employed as carriersfor the pharmaceutical compositions of this disclosure. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763,5,814,344 and 5,942,252.

The compound can be administered in liposomes or microspheres (ormicroparticles). Methods for preparing liposomes and microspheres foradministration to a subject are well known to those of skill in the art.U.S. Pat. No. 4,789,734, the contents of which are hereby incorporatedby reference, describes methods for encapsulating biological materialsin liposomes. Essentially, the material is dissolved in an aqueoussolution, the appropriate phospholipids and lipids added, and along withsurfactants if required, and the material dialyzed or sonicated, asnecessary. A review of known methods is provided by G. Gregoriadis,Chapter 14, “Liposomes,” Drug Carriers in Biology and Medicine, pp.2.sup.87-341 (Academic Press, 1979).

Microspheres formed of polymers or polypeptides are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contentsof which are hereby incorporated by reference.

The concentration of drug can be adjusted, the pH of the solutionbuffered and the isotonicity adjusted to be compatible with intravenousinjection, as is well known in the art.

The compounds of the disclosure can be formulated as a sterile solutionor suspension, in suitable vehicles, well known in the art. Thepharmaceutical compositions can be sterilized by conventional,well-known sterilization techniques, or can be sterile filtered. Theresulting aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. Suitable formulations and additionalcarriers are described in Remington “The Science and Practice ofPharmacy” (20th Ed., Lippincott Williams & Wilkins, Baltimore Md.), theteachings of which are incorporated by reference in their entiretyherein.

The agents or their pharmaceutically acceptable salts can be providedalone or in combination with one or more other agents or with one ormore other forms. For example, a formulation can comprise one or moreagents in particular proportions, depending on the relative potencies ofeach agent and the intended indication. For example, in compositions fortargeting two different host targets, and where potencies are similar,about a 1:1 ratio of agents can be used. The two forms can be formulatedtogether, in the same dosage unit e.g., in one cream, suppository,tablet, capsule, aerosol spray, or packet of powder to be dissolved in abeverage; or each form can be formulated in a separate unit, e.g., twocreams, two suppositories, two tablets, two capsules, a tablet and aliquid for dissolving the tablet, two aerosol sprays, or a packet ofpowder and a liquid for dissolving the powder, etc.

The term “pharmaceutically acceptable salt” means those salts whichretain the biological effectiveness and properties of the agents used inthe present disclosure, and which are not biologically or otherwiseundesirable. For example, a pharmaceutically acceptable salt does notinterfere with the beneficial effect of an agent of the disclosure ininhibiting a developmental disorder associated biomarkers' components

Typical salts are those of the inorganic ions, such as, for example,sodium, potassium, calcium, magnesium ions, and the like. Such saltsinclude salts with inorganic or organic acids, such as hydrochloricacid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid,methanesulfonic acid, p toluenesulfonic acid, acetic acid, fumaric acid,succinic acid, lactic acid, mandelic acid, malic acid, citric acid,tartaric acid or maleic acid. In addition, if the agent(s) contain acarboxy group or other acidic group, it can be converted into apharmaceutically acceptable addition salt with inorganic or organicbases. Examples of suitable bases include sodium hydroxide, potassiumhydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,diethanolamine, triethanolamine, and the like.

A pharmaceutically acceptable ester or amide refers to those whichretain biological effectiveness and properties of the agents used in thepresent disclosure, and which are not biologically or otherwiseundesirable. For example, the ester or amide does not interfere with thebeneficial effect of an agent of the disclosure in inhibiting adevelopmental disorder associated biomarkers' components. Typical estersinclude ethyl, methyl, isobutyl, ethylene glycol, and the like. Typicalamides include unsubstituted amides, alkyl amides, dialkyl amides, andthe like.

In some embodiments, an agent can be administered in combination withone or more other compounds, forms, and/or agents, e.g., as describedabove. Pharmaceutical compositions comprising combinations of adevelopmental disorder associated biomarkers' inhibitors with one ormore other active agents can be formulated to comprise certain molarratios. For example, molar ratios of about 99:1 to about 1:99 of adevelopmental disorder associated biomarkers' inhibitors to the otheractive agent can be used. In some subset of the embodiments, the rangeof molar ratios of developmental disorder associated biomarkers'inhibitors: other active agents are selected from about 80:20 to about20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about90:10 to about 10:90. The molar ratio of developmental disorderassociated biomarkers' inhibitors: other active agents can be about 1:9,and in some embodiments can be about 1:1. The two agents, forms and/orcompounds can be formulated together, in the same dosage unit e.g., inone cream, suppository, tablet, capsule, or packet of powder to bedissolved in a beverage; or each agent, form, and/or compound can beformulated in separate units, e.g., two creams, suppositories, tablets,two capsules, a tablet and a liquid for dissolving the tablet, anaerosol spray a packet of powder and a liquid for dissolving the powder,etc.

If necessary or desirable, the agents and/or combinations of agents canbe administered with still other agents. The choice of agents that canbe co-administered with the agents and/or combinations of agents of theinstant disclosure can depend, at least in part, on the condition beingtreated. Agents of particular use in the formulations of the presentdisclosure include, for example, any agent having a therapeutic effectfor a viral infection, including, e.g., drugs used to treat inflammatoryconditions. For example, in treatments for influenza, in someembodiments formulations of the instant disclosure can additionallycontain one or more conventional anti-inflammatory drugs, such as anNSAID, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin.In some alternative embodiments for the treatment of influenzaformulations of the instant disclosure can additionally contain one ormore conventional influenza antiviral agents, such as amantadine,rimantadine, zanamivir, and oseltamivir. In treatments for retroviralinfections, such as HIV, formulations of the instant disclosure canadditionally contain one or more conventional antiviral drug, such asprotease inhibitors (lopinavir/ritonavir {Kaletra}, indinavir{Crixivan}, ritonavir {Norvir}, nelfinavir {Viracept}, saquinavir hardgel capsules {Invirase}, atazanavir {Reyataz}, amprenavir {Agenerase},fosamprenavir {Telzir}, tipranavir{Aptivus}), reverse transcriptaseinhibitors, including non-Nucleoside and Nucleoside/nucleotideinhibitors (AZT {zidovudine, Retrovir}, ddl {didanosine, Videx}, 3TC{lamivudine, Epivir}, d4T {stavudine, Zerit}, abacavir {Ziagen}, FTC{emtricitabine, Emtriva}, tenofovir {Viread}, efavirenz {Sustiva} andnevirapine {Viramune}), fusion inhibitors T20 {enfuvirtide, Fuzeon},integrase inhibitors (MK-0518 and GS-9137), and maturation inhibitors(PA-457 {Bevirimat}). As another example, formulations can additionallycontain one or more supplements, such as vitamin C, E or otheranti-oxidants.

The agent(s) (or pharmaceutically acceptable salts, esters or amidesthereof) can be administered per se or in the form of a pharmaceuticalcomposition wherein the active agent(s) is in an admixture or mixturewith one or more pharmaceutically acceptable carriers. A pharmaceuticalcomposition, as used herein, can be any composition prepared foradministration to a subject. Pharmaceutical compositions for use inaccordance with the present disclosure can be formulated in conventionalmanner using one or more physiologically acceptable carriers, comprisingexcipients, diluents, and/or auxiliaries, e.g., which facilitateprocessing of the active agents into preparations that can beadministered. Proper formulation can depend at least in part upon theroute of administration chosen. The agent(s) useful in the presentdisclosure, or pharmaceutically acceptable salts, esters, or amidesthereof, can be delivered to a subject using a number of routes or modesof administration, including oral, buccal, topical, rectal, transdermal,transmucosal, subcutaneous, intravenous, and intramuscular applications,as well as by inhalation.

For oral administration, the agents can be formulated readily bycombining the active agent(s) with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the agents of the disclosureto be formulated as tablets, including chewable tablets, pills, dragees,capsules, lozenges, hard candy, liquids, gels, syrups, slurries,powders, suspensions, elixirs, wafers, and the like, for oral ingestionby a subject to be treated. Such formulations can comprisepharmaceutically acceptable carriers including solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents. Asolid carrier can be one or more substances which can also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material. In powders, the carrier generally is a finelydivided solid which is a mixture with the finely divided activecomponent. In tablets, the active component generally is mixed with thecarrier having the necessary binding capacity in suitable proportionsand compacted in the shape and size desired. The powders and tabletspreferably contain from about one (1) to about seventy (70) percent ofthe active compound. Suitable carriers include but are not limited tomagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Generally, the agents of the disclosure can be included at concentrationlevels ranging from about 0.5%, about 5%, about 10%, about 20%, or about30% to about 50%, about 60%, about 70%, about 80% or about 90% by weightof the total composition of oral dosage forms, in an amount sufficientto provide a desired unit of dosage.

Aqueous suspensions for oral use can contain agent(s) of this disclosurewith pharmaceutically acceptable excipients, such as a suspending agent(e.g., methyl cellulose), a wetting agent (e.g., lecithin, lysolecithinand/or a long-chain fatty alcohol), as well as coloring agents,preservatives, flavoring agents, and the like.

In some embodiments, oils or non-aqueous solvents can be used to bringthe agents into solution, due to, for example, the presence of largelipophilic moieties. Alternatively, emulsions, suspensions, or otherpreparations, for example, liposomal preparations, can be used. Withrespect to liposomal preparations, any known methods for preparingliposomes for treatment of a condition can be used. See, for example,Bangham et al., J. Mol. Biol. 23: 238-252 (1965) and Szoka et al., Proc.Natl Acad. Sci. USA 75: 4194-4198 (1978), incorporated herein byreference. Ligands can also be attached to the liposomes to direct thesecompositions to particular sites of action. Agents of this disclosurecan also be integrated into foodstuffs, e.g., cream cheese, butter,salad dressing, or ice cream to facilitate solubilization,administration, and/or compliance in certain subject populations.

Pharmaceutical preparations for oral use can be obtained as a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; flavoring elements, cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone(PVP). If desired, disintegrating agents can be added, such as the crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereofsuch as sodium alginate. The agents can also be formulated as asustained release preparation.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active agents.

Pharmaceutical preparations that can be used orally include push fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active agents can be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers can be added. All formulations fororal administration should be in dosages suitable for administration.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, or solid form preparations which are intended to beconverted shortly before use to liquid form preparations. Emulsions canbe prepared in solutions, for example, in aqueous propylene glycolsolutions or can contain emulsifying agents, for example, such aslecithin, sorbitan monooleate, or acacia. Aqueous solutions can beprepared by dissolving the active component in water and adding suitablecolorants, flavors, stabilizers, and thickening agents. Aqueoussuspensions can be prepared by dispersing the finely divided activecomponent in water with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and otherwell known suspending agents. Suitable fillers or carriers with whichthe compositions can be administered include agar, alcohol, fats,lactose, starch, cellulose derivatives, polysaccharides,polyvinylpyrrolidone, silica, sterile saline and the like, or mixturesthereof used in suitable amounts. Solid form preparations includesolutions, suspensions, and emulsions, and can contain, in addition tothe active component, colorants, flavors, stabilizers, buffers,artificial and natural sweeteners, dispersants, thickeners, solubilizingagents, and the like.

A syrup or suspension can be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which canalso be added any accessory ingredients. Such accessory ingredients caninclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

When formulating compounds of the disclosure for oral administration, itcan be desirable to utilize gastroretentive formulations to enhanceabsorption from the gastrointestinal (GI) tract. A formulation which isretained in the stomach for several hours can release compounds of thedisclosure slowly and provide a sustained release that can be preferredin some embodiments of the disclosure. Disclosure of suchgastro-retentive formulations are found in Klausner, E. A.; Lavy, E.;Barta, M.; Cserepes, E.; Friedman, M.; Hoffman, A. 2003 “Novelgastroretentive dosage forms: evaluation of gastroretentivity and itseffect on levodopa in humans.” Pharm. Res. 20, 1466-73, Hoffman, A.;Stepensky, D.; Lavy, E.; Eyal, S. Klausner, E.; Friedman, M. 2004“Pharmacokinetic and pharmacodynamic aspects of gastroretentive dosageforms” Int. J. Pharm. 11, 141-53, Streubel, A.; Siepmann, J.; Bodmeier,R.; 2006 “Gastroretentive drug delivery systems” Expert Opin. DrugDeliver. 3, 217-3, and Chavanpatil, M. D.; Jain, P.; Chaudhari, S.;Shear, R.; Vavia, P. R. “Novel sustained release, swellable andbioadhesive gastroretentive drug delivery system for olfoxacin” Int. J.Pharm. 2006. Expandable, floating and bioadhesive techniques can beutilized to maximize absorption of the compounds of the disclosure.

The compounds of the disclosure can be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and can be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions can take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, forexample, solutions in aqueous polyethylene glycol.

For injectable formulations, the vehicle can be chosen from those knownin art to be suitable, including aqueous solutions or oil suspensions,or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil,as well as elixirs, mannitol, dextrose, or a sterile aqueous solution,and similar pharmaceutical vehicles. The formulation can also comprisepolymer compositions which are biocompatible, biodegradable, such aspoly(lactic-co-glycolic)acid. These materials can be made into micro ornanospheres, loaded with drug and further coated or derivatized toprovide superior sustained release performance. Vehicles suitable forperiocular or intraocular injection include, for example, suspensions oftherapeutic agent in injection grade water, liposomes and vehiclessuitable for lipophilic substances. Other vehicles for periocular orintraocular injection are well known in the art.

In some embodiments, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition can also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

When administration is by injection, the active compound can beformulated in aqueous solutions, specifically in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiological saline buffer. The solution can contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,the active compound can be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use. In someembodiments, the pharmaceutical composition does not comprise anadjuvant or any other substance added to enhance the immune responsestimulated by the peptide. In some embodiments, the pharmaceuticalcomposition comprises a substance that inhibits an immune response tothe peptide. Methods of formulation are known in the art, for example,as disclosed in Remington's Pharmaceutical Sciences, latest edition,Mack Publishing Co., Easton P.

In addition to the formulations described previously, the agents canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation or transcutaneous delivery (forexample, subcutaneously or intramuscularly), intramuscular injection oruse of a transdermal patch. Thus, for example, the agents can beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

In some embodiments, pharmaceutical compositions comprising one or moreagents of the present disclosure exert local and regional effects whenadministered topically or injected at or near particular sites ofinfection. Direct topical application, e.g., of a viscous liquid,solution, suspension, dimethylsulfoxide (DMSO)-based solutions,liposomal formulations, gel, jelly, cream, lotion, ointment,suppository, foam, or aerosol spray, can be used for localadministration, to produce for example, local and/or regional effects.Pharmaceutically appropriate vehicles for such formulation include, forexample, lower aliphatic alcohols, polyglycols (e.g., glycerol orpolyethylene glycol), esters of fatty acids, oils, fats, silicones, andthe like. Such preparations can also include preservatives (e.g.,p-hydroxybenzoic acid esters) and/or antioxidants (e.g., ascorbic acidand tocopherol). See also Dermatological Formulations: Percutaneousabsorption, Barry (Ed.), Marcel Dekker Incl, 1983.

Pharmaceutical compositions of the present disclosure can contain acosmetically or dermatologically acceptable carrier. Such carriers arecompatible with skin, nails, mucous membranes, tissues and/or hair, andcan include any conventionally used cosmetic or dermatological carriermeeting these requirements. Such carriers can be readily selected by oneof ordinary skill in the art. In formulating skin ointments, an agent orcombination of agents of the instant disclosure can be formulated in anoleaginous hydrocarbon base, an anhydrous absorption base, awater-in-oil absorption base, an oil-in-water water-removable baseand/or a water-soluble base. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, water, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Ointments and creams can, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions can be formulated with an aqueous or oily base and canin general also containing one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches can beconstructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the disclosure include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

The compositions according to the present disclosure can be in any formsuitable for topical application, including aqueous, aqueous-alcoholicor oily solutions, lotion or serum dispersions, aqueous, anhydrous oroily gels, emulsions obtained by dispersion of a fatty phase in anaqueous phase (0/W or oil in water) or, conversely, (W/O or water inoil), microemulsions or alternatively microcapsules, microparticles orlipid vesicle dispersions of ionic and/or nonionic type. Thesecompositions can be prepared according to conventional methods. Otherthan the agents of the disclosure, the amounts of the variousconstituents of the compositions according to the disclosure are thoseconventionally used in the art. These compositions in particularconstitute protection, treatment or care creams, milks, lotions, gels orfoams for the face, for the hands, for the body and/or for the mucousmembranes, or for cleansing the skin. The compositions can also consistof solid preparations constituting soaps or cleansing bars.

Compositions of the present disclosure can also contain adjuvants commonto the cosmetic and dermatological fields, such as hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic active agents,preserving agents, antioxidants, solvents, fragrances, fillers,sunscreens, odor-absorbers and dyestuffs. The amounts of these variousadjuvants are those conventionally used in the fields considered and,for example, are from about 0.01% to about 20% of the total weight ofthe composition. Depending on their nature, these adjuvants can beintroduced into the fatty phase, into the aqueous phase and/or into thelipid vesicles.

In some embodiments, ocular viral infections can be effectively treatedwith ophthalmic solutions, suspensions, ointments or inserts comprisingan agent or combination of agents of the present disclosure. Eye dropscan be prepared by dissolving the active ingredient in a sterile aqueoussolution such as physiological saline, buffering solution, etc., or bycombining powder compositions to be dissolved before use. Other vehiclescan be chosen, as is known in the art, including but not limited to:balance salt solution, saline solution, water soluble polyethers such aspolyethyene glycol, polyvinyls, such as polyvinyl alcohol and povidone,cellulose derivatives such as methylcellulose and hydroxypropylmethylcellulose, petroleum derivatives such as mineral oil and whitepetrolatum, animal fats such as lanolin, polymers of acrylic acid suchas carboxypolymethylene gel, vegetable fats such as peanut oil andpolysaccharides such as dextrans, and glycosaminoglycans such as sodiumhyaluronate. If desired, additives ordinarily used in the eye drops canbe added. Such additives include isotonizing agents (e.g., sodiumchloride, etc.), buffer agent (e.g., boric acid, sodium monohydrogenphosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g.,benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.),thickeners (e.g., saccharide such as lactose, mannitol, maltose, etc.;e.g., hyaluronic acid or its salt such as sodium hyaluronate, potassiumhyaluronate, etc.; e.g., mucopolysaccharide such as chondroitin sulfate,etc.; e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinkedpolyacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, methylcellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose,carboxymethyl cellulose, hydroxy propyl cellulose or other agents knownto those skilled in the art).

The solubility of the components of the present compositions can beenhanced by a surfactant or other appropriate co-solvent in thecomposition. Such cosolvents include polysorbate 20, 60, and 80,Pluronic F68, F-84 and P-103, cyclodextrin, or other agents known tothose skilled in the art. Such co-solvents can be employed at a level offrom about 0.01% to 2% by weight.

The compositions of the disclosure can be packaged in multidose form.Preservatives can be preferred to prevent microbial contamination duringuse. Suitable preservatives include: benzalkonium chloride, thimerosal,chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol,edetate disodium, sorbic acid, Onamer M, or other agents known to thoseskilled in the art. In the prior art ophthalmic products, suchpreservatives can be employed at a level of from 0.004% to 0.02%. In thecompositions of the present application the preservative, preferablybenzalkonium chloride, can be employed at a level of from 0.001% to lessthan 0.01%, e.g. from 0.001% to 0.008%, preferably about 0.005% byweight. It has been found that a concentration of benzalkonium chlorideof 0.005% can be sufficient to preserve the compositions of the presentdisclosure from microbial attack.

In some embodiments, developmental disorder associated symptoms of theear can be effectively treated with otic solutions, suspensions,ointments or inserts comprising an agent or combination of agents of thepresent disclosure.

In some embodiments, the agents of the present disclosure are deliveredin soluble rather than suspension form, which allows for more rapid andquantitative absorption to the sites of action. In general, formulationssuch as jellies, creams, lotions, suppositories and ointments canprovide an area with more extended exposure to the agents of the presentdisclosure, while formulations in solution, e.g., sprays, provide moreimmediate, short-term exposure.

In some embodiments relating to topical/local application, thepharmaceutical compositions can include one or more penetrationenhancers. For example, the formulations can comprise suitable solid orgel phase carriers or excipients that increase penetration or helpdelivery of agents or combinations of agents of the disclosure across apermeability barrier, e.g., the skin. Many of thesepenetration-enhancing compounds are known in the art of topicalformulation, and include, e.g., water, alcohols (e.g., terpenes likemethanol, ethanol, 2-propanol), sulfoxides (e.g., dimethyl sulfoxide,decylmethyl sulfoxide, tetradecylmethyl sulfoxide), pyrrolidones (e.g.,2-pyrrolidone, N-methyl-2-pyrrolidone, N-(2-hydroxyethyl)pyrrolidone),laurocapram, acetone, dimethylacetamide, dimethylformamide,tetrahydrofurfuryl alcohol, L-a-amino acids, anionic, cationic,amphoteric or nonionic surfactants (e.g., isopropyl myristate and sodiumlauryl sulfate), fatty acids, fatty alcohols (e.g., oleic acid), amines,amides, clofibric acid amides, hexamethylene lauramide, proteolyticenzymes, a-bisabolol, d-limonene, urea and N,N-diethyl-m-toluamide, andthe like. Additional examples include humectants (e.g., urea), glycols(e.g., propylene glycol and polyethylene glycol), glycerol monolaurate,alkanes, alkanols, ORGELASE, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and/or otherpolymers. In some embodiments, the pharmaceutical compositions caninclude one or more such penetration enhancers.

In some embodiments, the pharmaceutical compositions for local/topicalapplication can include one or more antimicrobial preservatives such asquaternary ammonium compounds, organic mercurials, p-hydroxy benzoates,aromatic alcohols, chlorobutanol, and the like.

Gastrointestinal developmental disorder symptoms can be effectivelytreated with orally- or rectally delivered solutions, suspensions,ointments, enemas and/or suppositories comprising an agent orcombination of agents of the present disclosure.

Respiratory developmental disorder symptoms can be effectively treatedwith aerosol solutions, suspensions or dry powders comprising an agentor combination of agents of the present disclosure. Administration byinhalation is particularly useful in treating viral infections of thelung, such as influenza. The aerosol can be administered through therespiratory system or nasal passages. For example, one skilled in theart can recognize that a composition of the present disclosure can besuspended or dissolved in an appropriate carrier, e.g., apharmaceutically acceptable propellant, and administered directly intothe lungs using a nasal spray or inhalant For example, an aerosolformulation comprising a developmental disorder associated biomarkers'inhibitors can be dissolved, suspended or emulsified in a propellant ora mixture of solvent and propellant, e.g., for administration as a nasalspray or inhalant Aerosol formulations can contain any acceptablepropellant under pressure, such as a cosmetically or dermatologically orpharmaceutically acceptable propellant, as conventionally used in theart.

An aerosol formulation for nasal administration is generally an aqueoussolution designed to be administered to the nasal passages in drops orsprays. Nasal solutions can be similar to nasal secretions in that theyare generally isotonic and slightly buffered to maintain a pH of about5.5 to about 6.5, although pH values outside of this range canadditionally be used. Antimicrobial agents or preservatives can also beincluded in the formulation.

An aerosol formulation for inhalations and inhalants can be designed sothat the agent or combination of agents of the present disclosure iscarried into the respiratory tree of the subject when administered bythe nasal or oral respiratory route. Inhalation solutions can beadministered, for example, by a nebulizer. Inhalations or insufflations,comprising finely powdered or liquid drugs, can be delivered to therespiratory system as a pharmaceutical aerosol of a solution orsuspension of the agent or combination of agents in a propellant, e.g.,to aid in disbursement. Propellants can be liquefied gases, includinghalocarbons, for example, fluorocarbons such as fluorinated chlorinatedhydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as wellas hydrocarbons and hydrocarbon ethers.

Halocarbon propellants useful in the present disclosure includefluorocarbon propellants in which all hydrogens are replaced withfluorine, chlorofluorocarbon propellants in which all hydrogens arereplaced with chlorine and at least one fluorine, hydrogen-containingfluorocarbon propellants, and hydrogen-containing chlorofluorocarbonpropellants. Halocarbon propellants are described in Johnson, U.S. Pat.No. 5,376,359; Byron et al., U.S. Pat. No. 5,190,029; and Purewal etal., U.S. Pat. No. 5,776,434. Hydrocarbon propellants useful in thedisclosure include, for example, propane, isobutane, n-butane, pentane,isopentane and neopentane. A blend of hydrocarbons can also be used as apropellant. Ether propellants include, for example, dimethyl ether aswell as the ethers. An aerosol formulation of the disclosure can alsocomprise more than one propellant. For example, the aerosol formulationcan comprise more than one propellant from the same class, such as twoor more fluorocarbons; or more than one, more than two, more than threepropellants from different classes, such as a fluorohydrocarbon and ahydrocarbon. Pharmaceutical compositions of the present disclosure canalso be dispensed with a compressed gas, e.g., an inert gas such ascarbon dioxide, nitrous oxide or nitrogen.

Aerosol formulations can also include other components, for example,ethanol, isopropanol, propylene glycol, as well as surfactants or othercomponents such as oils and detergents. These components can serve tostabilize the formulation and/or lubricate valve components.

The aerosol formulation can be packaged under pressure and can beformulated as an aerosol using solutions, suspensions, emulsions,powders and semisolid preparations. For example, a solution aerosolformulation can comprise a solution of an agent of the disclosure suchas a developmental disorder associated biomarkers' inhibitors in(substantially) pure propellant or as a mixture of propellant andsolvent. The solvent can be used to dissolve the agent and/or retard theevaporation of the propellant. Solvents useful in the disclosureinclude, for example, water, ethanol and glycols. Any combination ofsuitable solvents can be use, optionally combined with preservatives,antioxidants, and/or other aerosol components.

An aerosol formulation can also be a dispersion or suspension. Asuspension aerosol formulation can comprise a suspension of an agent orcombination of agents of the instant disclosure, e.g., a developmentaldisorder associated biomarkers' inhibitors, and a dispersing agent.Dispersing agents useful in the disclosure include, for example,sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil. Asuspension aerosol formulation can also include lubricants,preservatives, antioxidant, and/or other aerosol components.

An aerosol formulation can similarly be formulated as an emulsion. Anemulsion aerosol formulation can include, for example, an alcohol suchas ethanol, a surfactant, water and a propellant, as well as an agent orcombination of agents of the disclosure, e.g., a developmental disorderassociated biomarkers' inhibitors. The surfactant used can be nonionic,anionic or cationic. One example of an emulsion aerosol formulationcomprises, for example, ethanol, surfactant, water and propellant.Another example of an emulsion aerosol formulation comprises, forexample, vegetable oil, glyceryl monostearate and propane.

The compounds of the disclosure can be formulated for administration assuppositories. A low melting wax, such as a mixture of triglycerides,fatty acid glycerides, Witepsol S55 (trademark of Dynamite NobelChemical, Germany), or cocoa butter is first melted and the activecomponent is dispersed homogeneously, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sized molds,allowed to cool, and to solidify.

The compounds of the disclosure can be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

It is envisioned additionally, that the compounds of the disclosure canbe attached releasably to biocompatible polymers for use in sustainedrelease formulations on, in or attached to inserts for topical,intraocular, periocular, or systemic administration. The controlledrelease from a biocompatible polymer can be utilized with a watersoluble polymer to form an instillable formulation, as well. Thecontrolled release from a biocompatible polymer, such as for example,PLGA microspheres or nanospheres, can be utilized in a formulationsuitable for intra ocular implantation or injection for sustainedrelease administration, as well any suitable biodegradable andbiocompatible polymer can be used.

In one aspect of the disclosure, the subject's carrier status of any ofthe genetic variation risk variants described herein, or geneticvariants identified via other analysis methods within the genes orregulatory loci that are identified by the CNVs described herein, can beused to help determine whether a particular treatment modality for adevelopmental disorder, such as any one of the above, or a combinationthereof, should be administered. The present disclosure also relates tomethods of monitoring progress or effectiveness of a treatment optionfor a developmental disorder. The treatment option can include any ofthe above mentioned treatment options commonly used. This can be donebased on the outcome of determination of the presence of a particulargenetic variation risk variant in the individual, or by monitoringexpression of genes that are associated with the variants of the presentdisclosure. Expression levels and/or mRNA levels can thus be determinedbefore and during treatment to monitor its effectiveness. Alternatively,or concomitantly, the status with respect to a genetic variation, and orgenotype and/or haplotype status of at least one risk variant for adevelopmental disorder presented herein can determined before and duringtreatment to monitor its effectiveness. It can also be appreciated bythose skilled in the art that aberrant expression levels of a geneimpacted by a CNV or other mutations found as a consequence of targetedsequencing of the CNV-identified gene can be assayed or diagnosticallytested for by measuring the polypeptide expression level of saidaberrantly expressed gene. In another embodiment, aberrant expressionlevels of a gene may result from a CNV impacting a DNA sequence (e.g.,transcription factor binding site) that regulates a gene who's aberrantexpression level is involved in or causes a developmental disorder, orother mutations found as a consequence of targeted sequencing of theCNV-identified gene regulatory sequence, can be assayed ordiagnostically tested for by measuring the polypeptide expression levelof the gene involved in or causative of a developmental disorder. Insome embodiments, a specific CNV mutation within a gene, or otherspecific mutations found upon targeted sequencing of a CNV-identifiedgene found to be involved in or causative of a developmental disorder,may cause an aberrant structural change in the expressed polypeptidethat results from said gene mutations and the altered polypeptidestructure(s) can be assayed via various methods know to those skilled inthe art.

Alternatively, biological networks or metabolic pathways related to thegenes within, or associated with, the genetic variations describedherein can be monitored by determining mRNA and/or polypeptide levels.This can be done for example, by monitoring expression levels ofpolypeptides for several genes belonging to the network and/or pathwayin nucleic acid samples taken before and during treatment.Alternatively, metabolites belonging to the biological network ormetabolic pathway can be determined before and during treatment.Effectiveness of the treatment is determined by comparing observedchanges in expression levels/metabolite levels during treatment tocorresponding data from healthy subjects.

In a further aspect, the genetic variations described herein and/orthose subsequently found (e.g., via other genetic analysis methods suchas sequencing) via targeted analysis of those genes initially identifiedby the genetic variations described herein, can be used to increasepower and effectiveness of clinical trials. Thus, individuals who arecarriers of at least one at-risk genetic variation can be more likely torespond to a particular treatment modality for a developmental disorder.In some embodiments, individuals who carry at-risk variants for gene(s)in a pathway and/or metabolic network for which a particular treatmentis targeting are more likely to be responders to the treatment. In someembodiments, individuals who carry at-risk variants for a gene, whichexpression and/or function is altered by the at-risk variant, are morelikely to be responders to a treatment modality targeting that gene, itsexpression or its gene product. This application can improve the safetyof clinical trials, but can also enhance the chance that a clinicaltrial can demonstrate statistically significant efficacy, which can belimited to a certain sub-group of the population. Thus, one possibleoutcome of such a trial is that carriers of certain genetic variants arestatistically significant and likely to show positive response to thetherapeutic agent. Further, one or more of the genetic variationsemployed during clinical trials for a given therapeutic agent can beused in a companion diagnostic test that is administered to the patientprior to administration of the therapeutic agent to determine if thepatient is likely to have favorable response to the therapeutic agent.

In a further aspect, the genetic variations described herein can be usedfor targeting the selection of pharmaceutical agents for specificindividuals. The pharmaceutical agent can be any of the agents describedin the above. Personalized selection of treatment modalities, lifestylechanges or combination of the two, can be realized by the utilization ofthe at-risk genetic variations or surrogate markers in linkagedisequilibrium with the genetic variations. Thus, the knowledge of anindividual's status for particular genetic variations can be useful forselection of treatment options, for example, for treatments that targetgenes or gene products affected by one or more of the geneticvariations. Certain combinations of variants, including those describedherein, but also combinations with other risk variants for adevelopmental disorder, can be suitable for one selection of treatmentoptions, while other variant combinations can target other treatmentoptions. Such combinations of variants can include one variant, twovariants, three variants, or four or more variants, as needed todetermine with clinically reliable accuracy the selection of treatmentmodule.

Animal and Cell Models of Developmental Disorders

Also provided herein are engineered cells that can harbor one or morepolymorphism described herein, for example, one or more geneticvariations associated with a developmental disorder, for example a SNPor CNV. Such cells can be useful for studying the effect of apolymorphism on physiological function, and for identifying and/orevaluating potential therapeutic agents.

Methods are known in the art for generating cells, for example, byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell, for example, a cell of an animal.In some cases, cells can be used to generate transgenic animals usingmethods known in the art.

The cells are preferably mammalian cells in which an endogenous gene hasbeen altered to include a genetic variation as described herein.Techniques such as targeted homologous recombination, can be used toinsert the heterologous DNA as described in, e.g., Chappel, U.S. Pat.No. 5,272,071; WO 91/06667. In another embodiment induced pluripotentstem cells with specific disease-causing or disease-associated mutations(such as CNVs and SNVs) can be used for disease modeling and drugdiscovery, for example, as described in Grskovic et al. (2011) Nat. Rev.Drug. Discov. 10(12):915-29.

Autism Spectrum Disorder is not known to occur naturally in any speciesother than humans, although animal models which show some features ofthe disease. This mouse model was created by replacing the normal mouseneurologin-3 gene with a mutated neuroligin-3 gene associated withautism in humans (Siidhof, M. D., et al., UT Southwestern). By doing so,a gene was created in mice similar to the human autism disease gene.While the result amounted to a very small change in their geneticmakeup, it mimics the same small change occurring in some patients withhuman autism. This and any other models described in the literature canbe used with the methods of the disclosure.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are present in aneffective amount, i.e., in an amount effective to achieve therapeuticand/or prophylactic benefit in a host with at least one a developmentaldisorder associated symptom. The actual amount effective for aparticular application can depend on the condition or conditions beingtreated, the condition of the subject, the formulation, and the route ofadministration, as well as other factors known to those of skill in theart. Determination of an effective amount of a developmental disorderassociated biomarkers' inhibitors is well within the capabilities ofthose skilled in the art, in light of the disclosure herein, and can bedetermined using routine optimization techniques.

The effective amount for use in humans can be determined from animalmodels. For example, a dose for humans can be formulated to achievecirculating, liver, topical and/or gastrointestinal concentrations thathave been found to be effective in animals. One skilled in the art candetermine the effective amount for human use, especially in light of theanimal model experimental data described herein. Based on animal data,and other types of similar data, those skilled in the art can determinethe effective amounts of compositions of the present disclosureappropriate for humans.

The effective amount when referring to an agent or combination of agentsof the disclosure can generally mean the dose ranges, modes ofadministration, formulations, etc., that have been recommended orapproved by any of the various regulatory or advisory organizations inthe medical or pharmaceutical arts (e.g., FDA, AMA) or by themanufacturer or supplier.

Further, appropriate doses for a developmental disorder associatedbiomarkers' inhibitors can be determined based on in vitro experimentalresults. For example, the in vitro potency of an agent in inhibiting adevelopmental disorder associated biomarkers' components, providesinformation useful in the development of effective in vivo dosages toachieve similar biological effects. In some embodiments, administrationof agents of the present disclosure can be intermittent, for example,administration once every two days, every three days, every five days,once a week, once or twice a month, and the like. In some embodiments,the amount, forms, and/or amounts of the different forms can be variedat different times of administration.

A person of skill in the art would be able to monitor in a subject theeffect of administration of a particular agent. Other techniques wouldbe apparent to one of skill in the art, wherein the active ingredientsare present in an effective amount, for example, in an amount effectiveto achieve therapeutic and/or prophylactic benefit in a host with atleast one a developmental disorder associated symptom. The actual amounteffective for a particular application can depend on the condition orconditions being treated, the condition of the subject, the formulation,and the route of administration, as well as other factors known to thoseof skill in the art. Determination of an effective amount of adevelopmental disorder associated biomarkers' inhibitors is well withinthe capabilities of those skilled in the art, in light of the disclosureherein, and can be determined using routine optimization techniques.

Further, appropriate doses for a developmental disorder associatedbiomarkers' inhibitors can be determined based on in vitro experimentalresults. For example, the in vitro potency of an agent in inhibiting adevelopmental disorder associated biomarkers' components can provideinformation useful in the development of effective in vivo dosages toachieve similar biological effects.

Kits

Kits useful in the methods of the disclosure comprise components usefulin any of the methods described herein, including for example, primersfor nucleic acid amplification, hybridization probes for detectinggenetic variation, or other marker detection, restriction enzymes,nucleic acid probes, optionally labeled with suitable labels,allele-specific oligonucleotides, antibodies that bind to an alteredpolypeptide encoded by a nucleic acid of the disclosure as describedherein or to a wild type polypeptide encoded by a nucleic acid of thedisclosure as described herein, means for amplification of geneticvariations or fragments thereof, means for analyzing the nucleic acidsequence of nucleic acids comprising genetic variations as describedherein, means for analyzing the amino acid sequence of a polypeptideencoded by a genetic variation, or a nucleic acid associated with agenetic variation, etc. The kits can for example, include necessarybuffers, nucleic acid primers for amplifying nucleic acids, and reagentsfor allele-specific detection of the fragments amplified using suchprimers and necessary enzymes (e.g., DNA polymerase). Additionally, kitscan provide reagents for assays to be used in combination with themethods of the present disclosure, for example, reagents for use withother screening assays for a developmental disorder.

In some embodiments, the disclosure pertains to a kit for assaying anucleic acid sample from a subject to detect the presence of a geneticvariation, wherein the kit comprises reagents necessary for selectivelydetecting at least one particular genetic variation in the genome of theindividual. In some embodiments, the disclosure pertains to a kit forassaying a nucleic acid sample from a subject to detect the presence ofat least particular allele of at least one polymorphism associated witha genetic variation in the genome of the subject. In some embodiments,the reagents comprise at least one contiguous oligonucleotide thathybridizes to a fragment of the genome of the individual comprising atleast genetic variation. In some embodiments, the reagents comprise atleast one pair of oligonucleotides that hybridize to opposite strands ofa genomic segment obtained from a subject, wherein each oligonucleotideprimer pair is designed to selectively amplify a fragment of the genomeof the individual that includes at least one genetic variation, or afragment of a genetic variation. Such oligonucleotides or nucleic acidscan be designed using the methods described herein. In some embodiments,the kit comprises one or more labeled nucleic acids capable ofallele-specific detection of one or more specific polymorphic markers orhaplotypes with a genetic variation, and reagents for detection of thelabel. In some embodiments, a kit for detecting SNP markers can comprisea detection oligonucleotide probe, that hybridizes to a segment oftemplate DNA containing a SNP polymorphisms to be detected, an enhanceroligonucleotide probe, detection probe, primer and/or an endonuclease,for example, as described by Kutyavin et al. (Nucleic Acid Res. 34:e128(2006)).

In some embodiments, the DNA template is amplified by any means of thepresent disclosure, prior to assessment for the presence of specificgenetic variations as described herein. Standard methods well known tothe skilled person for performing these methods can be utilized, and arewithin scope of the disclosure. In one such embodiment, reagents forperforming these methods can be included in the reagent kit.

In a further aspect of the present disclosure, a pharmaceutical pack(kit) is provided, the pack comprising a therapeutic agent and a set ofinstructions for administration of the therapeutic agent to humansscreened for one or more variants of the present disclosure, asdisclosed herein. The therapeutic agent can be a small molecule drug, anantibody, a peptide, an antisense or RNAi molecule, or other therapeuticmolecules as described herein. In some embodiments, an individualidentified as a carrier of at least one variant of the presentdisclosure is instructed to take a prescribed dose of the therapeuticagent. In one such embodiment, an individual identified as a carrier ofat least one variant of the present disclosure is instructed to take aprescribed dose of the therapeutic agent. In some embodiments, anindividual identified as a non-carrier of at least one variant of thepresent disclosure is instructed to take a prescribed dose of thetherapeutic agent.

Also provided herein are articles of manufacture, comprising a probethat hybridizes with a region of human chromosome as described hereinand can be used to detect a polymorphism described herein. For example,any of the probes for detecting polymorphisms described herein can becombined with packaging material to generate articles of manufacture orkits. The kit can include one or more other elements including:instructions for use; and other reagents such as a label or an agentuseful for attaching a label to the probe. Instructions for use caninclude instructions for screening applications of the probe for makinga diagnosis, prognosis, or theranosis to a developmental disorder in amethod described herein. Other instructions can include instructions forattaching a label to the probe, instructions for performing in situanalysis with the probe, and/or instructions for obtaining a nucleicacid sample to be analyzed from a subject. In some cases, the kit caninclude a labeled probe that hybridizes to a region of human chromosomeas described herein.

The kit can also include one or more additional reference or controlprobes that hybridize to the same chromosome or another chromosome orportion thereof that can have an abnormality associated with aparticular endophenotype. A kit that includes additional probes canfurther include labels, e.g., one or more of the same or differentlabels for the probes. In other embodiments, the additional probe orprobes provided with the kit can be a labeled probe or probes. When thekit further includes one or more additional probe or probes, the kit canfurther provide instructions for the use of the additional probe orprobes. Kits for use in self-testing can also be provided. Such testkits can include devices and instructions that a subject can use toobtain a nucleic acid sample (e.g., buccal cells, blood) without the aidof a health care provider. For example, buccal cells can be obtainedusing a buccal swab or brush, or using mouthwash.

Kits as provided herein can also include a mailer (e.g., a postage paidenvelope or mailing pack) that can be used to return the nucleic acidsample for analysis, e.g., to a laboratory. The kit can include one ormore containers for the nucleic acid sample, or the nucleic acid samplecan be in a standard blood collection vial. The kit can also include oneor more of an informed consent form, a test requisition form, andinstructions on how to use the kit in a method described herein. Methodsfor using such kits are also included herein. One or more of the forms(e.g., the test requisition form) and the container holding the nucleicacid sample can be coded, for example, with a bar code for identifyingthe subject who provided the nucleic acid sample.

In some embodiments, an in vitro screening test can comprise one or moredevices, tools, and equipment configured to collect a nucleic acidsample from an individual. In some embodiments of an in vitro screeningtest, tools to collect a nucleic acid sample can include one or more ofa swab, a scalpel, a syringe, a scraper, a container, and other devicesand reagents designed to facilitate the collection, storage, andtransport of a nucleic acid sample. In some embodiments, an in vitroscreening test can include reagents or solutions for collecting,stabilizing, storing, and processing a nucleic acid sample.

Such reagents and solutions for nucleotide collecting, stabilizing,storing, and processing are well known by those of skill in the art andcan be indicated by specific methods used by an in vitro screening testas described herein. In some embodiments, an in vitro screening test asdisclosed herein, can comprise a microarray apparatus and reagents, aflow cell apparatus and reagents, a multiplex nucleotide sequencer andreagents, and additional hardware and software necessary to assay anucleic acid sample for certain genetic markers and to detect andvisualize certain genetic markers.

The present disclosure further relates to kits for using antibodies inthe methods described herein. This includes, but is not limited to, kitsfor detecting the presence of a variant polypeptide in a test nucleicacid sample. One preferred embodiment comprises antibodies such as alabeled or labelable antibody and a compound or agent for detectingvariant polypeptides in a nucleic acid sample, means for determining theamount or the presence and/or absence of variant polypeptide in thenucleic acid sample, and means for comparing the amount of variantpolypeptide in the nucleic acid sample with a standard, as well asinstructions for use of the kit. In certain embodiments, the kit furthercomprises a set of instructions for using the reagents comprising thekit.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The followingreferences contain embodiments of the methods and compositions that canbe used herein: The Merck Manual of Diagnosis and Therapy, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007(ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnol-ogy: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Standard procedures of the present disclosure are described, e.g., inManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrooket al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl (eds.),Academic Press Inc., San Diego, USA (1987)). Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols inImmunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manualof Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), and Animal Cell Culture Methods (Methods in CellBiology, Vol. 57, Jennie P. Mather and David Barnes editors, AcademicPress, 1st edition, 1998), which are all incorporated by referenceherein in their entireties.

It should be understood that the following examples should not beconstrued as being limiting to the particular methodology, protocols,and compositions, etc., described herein and, as such, can vary. Thefollowing terms used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of theembodiments disclosed herein.

Disclosed herein are molecules, materials, compositions, and componentsthat can be used for, can be used in conjunction with, can be used inpreparation for, or are products of methods and compositions disclosedherein. It is understood that when combinations, subsets, interactions,groups, etc. of these materials are disclosed and while specificreference of each various individual and collective combinations andpermutation of these molecules and compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a nucleotide or nucleic acid is disclosed and discussed anda number of modifications that can be made to a number of moleculesincluding the nucleotide or nucleic acid are discussed, each and everycombination and permutation of nucleotide or nucleic acid and themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed molecules and compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

Those skilled in the art can recognize, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagents describedas these can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present disclosure which canbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the meanings that would be commonly understood by one of skill inthe art in the context of the present specification.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleotide” includes a plurality of such nucleotides; reference to “thenucleotide” is a reference to one or more nucleotides and equivalentsthereof known to those skilled in the art, and so forth.

The term “and/or” shall in the present context be understood to indicatethat either or both of the items connected by it are involved. Whilepreferred embodiments of the present disclosure have been shown anddescribed herein, it can be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions can now occur to those skilled inthe art without departing from the disclosure. It should be understoodthat various alternatives to the embodiments of the disclosure describedherein can be employed in practicing the disclosure. It is intended thatthe following claims define the scope of the disclosure and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1

In the present study, the Agilent 1M CGH array was used to detect novel,rare CNVs in a total of 1687 individuals in 2 cohorts:

1. 1,005 Normal individuals (Normal Variation Engine—NVE);

2. 682 ASD cases (ASD).

The Normal DNA samples were from apparently healthy Caucasian donors >45years old. Health history information was documented at the time ofconsent via a questionnaire filled out by the donor. This informationwas used to select 1,000 Normals based on the following attributes: BMIbetween 15-35, blood glucose level <125 mg/dL, total cholesterol levelbetween 100-300, systolic blood pressure between 100-150, and no majorneurodegenerative diseases or psychiatric disorders (alcoholism, mentalillness, depression, dementia, Alzheimer's disease, and Parkinson'sdisease).

For the ASD samples, Reference DNA samples were labeled with Cy3 andtest subject cases with Cy5. After labeling, samples were combined andco-hybridized to Agilent 1M feature oligonucleotide microarrays, designID 021529 (Agilent Product Number G4447A) using standard conditions(array Comparative Genomic Hybridization—aCGH). Post-hybridization,arrays were scanned at 3 μm resolution, using Agilent's DNA microarrayscanner, generating tiff images for later analysis. All hybridizationswere sex-matched; reference samples were pools of 50 male and 50 femalesamples, respectively. Genomic DNA for the reference pools was isolatedfrom cell lines.

Genomic DNA samples from individuals within the Normal cohort (‘test’subjects) were hybridized against a single, sex-matched referenceindividual as follows. Reference DNA samples were labeled with Cy5 andTest subject DNA samples were labeled with Cy3. After labeling, sampleswere combined and co-hybridized to Agilent 1M feature oligonucleotidemicroarrays, design ID 021529 (Agilent Product Number G4447A) usingstandard conditions (array Comparative Genomic Hybridization—aCGH).Post-hybridization, arrays were scanned at 2 μm resolution, usingAgilent's DNA microarray scanner, generating tiff images for lateranalysis.

All tiff images were analyzed using Agilent Feature Extraction (FE)software, with the following settings:

Human Genome Freeze: hg18:NCBI36:Mar2006

FE version: 10.7.3.1

Grid/design file: 021529_D_F_20091001

Protocol: CGH_107_Sep09

This procedure generated a variety of output files, one of which was atext-tab delimited file, containing ˜1,000,000 rows of data, eachcorresponding to a specific feature on the array. This *.txt file wasused to perform CNV calling using DNAcopy, an open source softwarepackage implemented in R via BioConductor. Losses or gains weredetermined according to a threshold log 2ratio, which was set at−/+0.35. In other words, all losses with a log 2ratio value <−0.35 werecounted, as were all gains with a log 2ratio ≥+0.35. Log2ratio valueswere determined according to Cy3/Cy5 (Test/Reference). The minimum probenumber to call a CNV was set at 2 (2 consecutive probes were sufficientto call a CNV). A CNV list was thus generated for each individual in the2 cohorts.

There were a total of 162,316 CNVs in the NVE cohort of 1,005individuals. Using custom scripts, these CNVs (many of which appeared inmultiple individuals) were ‘merged’ into a master list (NVE-master) ofnon-redundant CNV-subregions, according to the presence or absence ofthe CNV-subregion in individuals within the cohort. Using this approach,the NVE-master list has 14,693 distinct CNV-subregions, some of whichare uniquely present in a single individual and some of which arepresent in multiple individuals. For example, consider 3 individualswithin the NVE cohort with the following hypothetical CNVs:

Chr1:1-100,000;

Chr1:10,001-100,000;

Chr1:1-89,999;

In the master list, these would be merged into 3 distinct CNVsubregions, as follows:

CNV-subregion 1 Chr1: 1-10,000 Patients A, C CNV-subregion 2 Chr1:10,001-89,999 Patients A, B, C CNV-subregion 3 Chr 90,000: 1-100,000Patients A, B

There were a total of 72,183 CNVs in the ASD cohort of 682 individuals.Using custom scripts, these CNVs (many of which appeared in multipleindividuals) were ‘merged’ into a master list (ASD-master) ofnon-redundant CNV-subregions, according to the presence or absence ofthe CNV-subregion in individuals within the cohort. Using this approach,the ASD-master list has 13,914 distinct CNV-subregions, some of whichare uniquely present in a single individual and some of which arepresent in multiple individuals.

CNV-subregions of interest were obtained after:

1. Annotation using custom designed scripts in order to attach to eachCNV region relevant information regarding overlap with known genes,exons and CNVs generated by a study from the Sanger institute(www.sanger.ac.uk/research/areas/humangenetics/cny/highres_discovery)

2. A calculation of the odds ratio (OR) for each CNV-subregion.

The OR for each subregion was calculated according to the followingformula:OR=(ASD/(682−ASD))/(NVE/(1005−NVE))where: ASD=number of ASD individuals with CNV-subregion of interest, andNVE=number of NVE individuals with CNV-subregion of interest

As an illustrative example, consider the CNV subregion chr1:750052-770858 (first row of Table 2), which is found in 1 individual inthe NVE cohort and 6 individuals in the ASD cohort.

The OR calculated was (61(682-6))/(1/(1005-1))=8.91.

By convention, if NVE=0, it is set to 1, in order to avoid dealing withinfinities. This has the effect of artificially lowering OR values incases where none are seen in the NVE. When calculating OR values foridentical CNV-subregions, gains and losses are combined.

CNV-subregions/genes that fulfill one of the following criteria weredetermined:

1. Genic (distinct CNV-subregions); OR >6

2. Exon+ve, ASD >4, NVE <2, no Sanger filter applied

3. Exon+ve, 5>ASD >1, Normals <2, Sanger filter −ve

4. Intron+ve, ASD >4, Normals <2, no Sanger filter applied

5. MTRNR2L family

6. High OR intergenic (OR >30)

The number of ASD candidate CNV-subregions, irrespective of category(genic or non-genic), may increase or decrease as additional ASD cohortsare analyzed A variety of CNVs may cause a pathogenic effect in affectedpatients that have at least one CNV from one of these categories. Forexample, CNVs can be non-overlapping (distinct CNVs) but all impact thesame gene (category 1). In other patients with a neurodevelopmentdisorder, the CNVs may be overlapping and/or non-overlapping, impact anexon, and they affect 5 or more cases but only 0 or 1 Normal subjectsand no filter of Sanger CNVs is applied due to the relatively high ORvalue (>7) for this category. In category 3, the CNVs may be overlappingand/or non-overlapping, impact an exon, and they affect <5 cases butonly 0 or 1 Normal subjects and no Sanger CNVs overlap, which enablesidentification of rarer CNVs in cases with a neurodevelopmental disorderbut with the stringency of Sanger CNVs that are presumed to berelatively common in the general population. Category 4 is equivalent tocategory 2 except that the CNVs impact an intron as it is appreciated bythose skilled in the art that genetic variants (such as CNVs) impactingintrons can be pathogenic (e.g., such variants can result inalternatively spliced mRNAs or loss of a microRNA binding site, whichmay deleteriously impact the resulting protein's structure or expressionlevel). Category 5 corresponds to CNVs that impact the MTRNR2L genefamily, which are also known as humanins (Matsuoka M, et al. Humanin andthe receptors for humanin. Mol Neurobiol. 2010 February; 41(1):22-8;Bodzioch M, et al. Evidence for potential functionality ofnuclearly-encoded humanin isoforms. Genomics. 2009 October;94(4):247-56; Maftei M, et al. Interaction structure of the complexbetween neuroprotective factor humanin and Alzheimer's (3-amyloidpeptide revealed by affinity mass spectrometry and molecular modeling. JPept Sci. 2012 June; 18(6):373-82; Arakawa T, et al. Advances incharacterization of neuroprotective peptide, humanin. Curr Med Chem.2011; 18(36):5554-63; Zapala B, et al. Humanins, the neuroprotective andcytoprotective peptides with antiapoptotic and anti-inflammatoryproperties. Pharmacol Rep. 2010 September-October; 62(5):767-77.

While humanins may have neuroprotective properties for Alzheimer'sdisease, it is not established in neurodevelopment disorders; however,recently links have been established between the Alzheimer's gene APPand neurodevelopmental disorders such as autism (Westmark CJ. What'shAPPening at synapses? The role of amyloid β-protein precursor andβ-amyloid in neurological disorders. Mol Psychiatry. 2012 Aug. 28).Category 6 CNVs are those that occur within intergenic regions but withhigh OR (>30) as it is well known by those skilled in the art that generegulatory regions often reside in adjacent regions of genes such ashave been experimentally determined and annotated in the ENCODE project(ENCODE Project Consortium, Bernstein B E, et al. An integratedencyclopedia of DNA elements in the human genome. Nature. 2012 Sep. 6;489(7414):57-74).

Example 2

Some pathway analysis software will be used to identify whether thecandidate gene will be a drug target, which may be FDA-approved or inclinical trials. Such information will assist in the design of clinicaltrials (e.g., patient stratification for genetic subtypes) or will beused to facilitate clinical trials that are in progress, therebyreducing the attrition rate (failure to receive FDA approval) andreducing the time and cost of drug development. When a candidate ASDgene is identified as a known drug target of an FDA-approvedtherapeutic, the drug can be repurposed and approved for use in a newindication (e.g., a cancer or anti-inflammatory agent may be beneficialto ASD patients as well). Those skilled in the art will recognize thatPhase II and III failures may be rescued with additional clinical trialdata that accounts for genetic subtypes, particularly when the drugfails for lack of efficacy. For example, if a drug will be designed orestablished to target a particular gene defect (e.g., use of an RNAitherapeutic to decrease aberrant overexpression of the gene that iscaused by a CNV or other type of genetic variant), it will be expectedthat only ASD patients with that particular genetic subtype will benefitfrom the targeted therapy.

Example 3

FIG. 1 represents an example of group 1 (Genic (distinctCNV-subregions); OR >6). There are 10 ASD cases and 0 NVE subjectsaffected by non-overlapping and overlapping CNV-subregions. The CNV aregains (log 2ratio >0.35) or losses (log 2ratio <−0.35) and affect thegene NRG1 on chromosome 8. The calculated odds ratio (OR) for thisCNV-subregion is 14.94.

In the figure, three tracks of information are shown, from top tobottom: 1) RefSeq gene annotation showing the genome location (x-axis)of genes demarcated in light gray (introns) and dark gray (exons) andwith multiple entries depicted if multiple transcript variants areannotated that correspond to the gene; 2) size and genome location(x-axis) for normal CNVs annotated for greater than 1,000unaffected/normal individuals, with CNVs demarcated by dark gray barsand the y-axis corresponds to the number of individuals in the normalcohort found to have the CNV; 3) array CGH data (black dots correspondto the probes on the microarray) for an ASD case with a CNV wherein they-axis is the log 2ratio value of the test (ASD case) and reference(healthy control) genomic DNAs and the x-axis corresponds to the genomelocation of the probes and CNVs, which are depicted as line segmentsshifted positively (copy number gain) or negatively (copy number loss)relative to the baseline (log 2 ratio=0).

Example 4

FIG. 2 represents an example of group 2 (Exon+ve, ASD >4, Normals <2, noSanger filter applied). There are 34 ASD cases in total (31 with anidentical loss) and 1 NVE subject affected by overlapping CNV-subregionsthat impact an exon. The CNV are a gain (log 2ratio >0.35) or losses(log 2ratio <−0.35) and affect the gene MIDN on chromosome 19. Thecalculated odds ratio (OR) for this CNV-subregion is 52.68.

In the figure, three tracks of information are shown, from top tobottom: 1) RefSeq gene annotation showing the genome location (x-axis)of genes demarcated in light gray (introns) and dark gray (exons) andwith multiple entries depicted if multiple transcript variants areannotated that correspond to the gene; 2) size and genome location(x-axis) for normal CNVs annotated for greater than 1,000unaffected/normal individuals, with CNVs demarcated by dark gray barsand the y-axis corresponds to the number of individuals in the normalcohort found to have the CNV; 3) array CGH data (black dots correspondto the probes on the microarray) for an ASD case with a CNV wherein they-axis is the log 2ratio value of the test (ASD case) and reference(healthy control) genomic DNAs and the x-axis corresponds to the genomelocation of the probes and CNVs, which are depicted as line segmentsshifted positively (copy number gain) or negatively (copy number loss)relative to the baseline (log 2 ratio=0).

Example 5

FIG. 3 represents an example of group 3 (Exon+ve, 5>ASD >1, Normals <2,Sanger −ve). There are 4 ASD cases in total and 1 NVE subject affectedby an overlapping CNV-subregion that impacts an exon. The CNV are losses(log 2ratio <−0.35) and affect the gene PTGER3 on chromosome 1 and noSanger CNVs overlap this CNV (Sanger −ve). The calculated odds ratio(OR) for this CNV-subregion is 5.92.

In the figure, three tracks of information are shown, from top tobottom: 1) RefSeq gene annotation showing the genome location (x-axis)of genes demarcated in light gray (introns) and dark gray (exons) andwith multiple entries depicted if multiple transcript variants areannotated that correspond to the gene; 2) size and genome location(x-axis) for normal CNVs annotated for greater than 1,000unaffected/normal individuals, with CNVs demarcated by dark gray barsand the y-axis corresponds to the number of individuals in the normalcohort found to have the CNV; 3) array CGH data (black dots correspondto the probes on the microarray) for an ASD case with a CNV wherein they-axis is the log 2ratio value of the test (ASD case) and reference(healthy control) genomic DNAs and the x-axis corresponds to the genomelocation of the probes and CNVs, which are depicted as line segmentsshifted positively (copy number gain) or negatively (copy number loss)relative to the baseline (log 2 ratio=0).

Example 6

FIG. 4 represents an example of group 4 (Intron+ve, ASD >4, Normals <2,no Sanger filter applied). There are 8 ASD cases in total (3 casesimpact an identical CNV loss) and 0 NVE subjects affected by anoverlapping CNV-subregion that impacts an intron. The CNV are losses(log 2ratio <−0.35) or a gain (log 2ratio >0.35) and affect the geneCALN1 on chromosome 7. The calculated odds ratio (OR) for thisCNV-subregion is 11.92.

In the figure, three tracks of information are shown, from top tobottom: 1) RefSeq gene annotation showing the genome location (x-axis)of genes demarcated in light gray (introns) and dark gray (exons) andwith multiple entries depicted if multiple transcript variants areannotated that correspond to the gene; 2) size and genome location(x-axis) for normal CNVs annotated for greater than 1,000unaffected/normal individuals, with CNVs demarcated by dark gray barsand the y-axis corresponds to the number of individuals in the normalcohort found to have the CNV; 3) array CGH data (black dots correspondto the probes on the microarray) for an ASD case with a CNV wherein they-axis is the log 2ratio value of the test (ASD case) and reference(healthy control) genomic DNAs and the x-axis corresponds to the genomelocation of the probes and CNVs, which are depicted as line segmentsshifted positively (copy number gain) or negatively (copy number loss)relative to the baseline (log 2 ratio=0).

Example 7

FIG. 5 represents an example of group 5 (MTRNR2L_family). There is 1 ASDcase and 0 NVE subjects that impact an exon of an MTRNR2L gene familymember. The CNV gain (log 2ratio >0.35) is 1.7 Mb in size and its leftbreakpoint disrupts MTRNR2L4 and its right breakpoint disrupts ALG1 onchromosome 16. The calculated odds ratio (OR) for this CNV-subregion is1.47.

The top panel shows the complete CNV, which impacts several genes. Thelower left panel is an expanded view of the left breakpoint depictingdisruption of MTRNR2L4. The lower right panel is an expanded view of theright breakpoint depicting disruption of ALG1. One or both genes may becausative of the patient's autistic phenotype.

In the figure, three tracks of information are shown, from top tobottom: 1) RefSeq gene annotation showing the genome location (x-axis)of genes demarcated in light gray (introns) and dark gray (exons) andwith multiple entries depicted if multiple transcript variants areannotated that correspond to the gene; 2) size and genome location(x-axis) for normal CNVs annotated for greater than 1,000unaffected/normal individuals, with CNVs demarcated by dark gray barsand the y-axis corresponds to the number of individuals in the normalcohort found to have the CNV; 3) array CGH data (black dots correspondto the probes on the microarray) for an ASD case with a CNV wherein they-axis is the log 2ratio value of the test (ASD case) and reference(healthy control) genomic DNAs and the x-axis corresponds to the genomelocation of the probes and CNVs, which are depicted as line segmentsshifted positively (copy number gain) or negatively (copy number loss)relative to the baseline (log 2 ratio=0).

Example 8

FIG. 6 represents an example of group 6 (High OR intergenic (OR >30)).There are 20 ASD cases in total (5 representative cases are depicted)and 0 NVE subjects affected by an overlapping CNV-subregion that impactsan intergenic region (adjacent to SDC1). The CNV are losses (log 2ratio<−0.35) on chromosome 2. The calculated odds ratio (OR) for thisCNV-subregion is 30.33.

In the figure, three tracks of information are shown, from top tobottom: 1) RefSeq gene annotation showing the genome location (x-axis)of genes demarcated in light gray (introns) and dark gray (exons) andwith multiple entries depicted if multiple transcript variants areannotated that correspond to the gene; 2) size and genome location(x-axis) for normal CNVs annotated for greater than 1,000unaffected/normal individuals, with CNVs demarcated by dark gray barsand the y-axis corresponds to the number of individuals in the normalcohort found to have the CNV; 3) array CGH data (black dots correspondto the probes on the microarray) for an ASD case with a CNV wherein they-axis is the log 2ratio value of the test (ASD case) and reference(healthy control) genomic DNAs and the x-axis corresponds to the genomelocation of the probes and CNVs, which are depicted as line segmentsshifted positively (copy number gain) or negatively (copy number loss)relative to the baseline (log 2 ratio=0).

Example 9 Example of Sequence Data

The sequence file ASD_ST25.txt contains genomic sequence information for(in the following order):

A. All distinct CNVs listed in Table 1 (SEQ_IDs 1-883);

The full genomic extent of the transcripts listed in Table 4 (SEQ_IDs884-1,690); Example of sequences submitted:

Sequence entry starts:SEQ_ID 1 = 1,337 bp loss/gain in an exon of MIDN, as listed in Table 1: <210> 1 <211> 1337 <212> DNA <213> Homo sapiens <400> 1ggaacgttga tacattataa cttttttttc ttgttacttt cacccccaga tcctccgagc   60ggcggcgacg gctgttgcta agggagggga cgcgcgagga agcgcgaccc gggcggcaga  120cggcacccag cgccaccagc cgagcggcgc cccctcccca ggacccttaa ccgcgccgcg  180tcccggtcgc gcccgccgcc ctttgaagga gaagcaagtg ccgtccccac ccccggaagg  240cgcccccagg agccggagcg acctcggagc gccactcgga ttttggattt cggtctcgca  300ttccgcggcc gggactttct cgaggaggac gcgcgctgct ccgcgccccc gagtgcccgg  360aggacccggc atccggggag cctctcgccc ctgtcccgga ggcgcggcga ggattggcgg  420cgcccgccgc ccccagcccc ccagcgcgcg ccggggatgg agccgcagcc cggcggcgcc  480cggagctgcc ggcgcggggc ccccggcggc gcctgcgagc tgggcccggc ggccgaggcg  540gcgcccatga gcctcgccat ccacagcacc acgggcaccc gctacgacct ggccgtgccg  600cccgacgaga cggtggaggg gctgcgcaag cggttgtccc agcgcctcaa agtgcccaag  660gagcgcctgg ctcttctcca caaagacacg taggtaccgc gcgcccccgg ccggccgccc  720cctcgggccc cggcccccgg gcgggaacaa agagcgcgcc gcgcggggaa ggcagggggc  780ggccagacag ggggcggggg cgcgccgcgc gctctcgggc gccctctgct cggcctcgcc  840tgcctcggcc ccctcccccg cccggggtcg ccgcacaaag gcggctgcga gggcgtcccg  900ggccgggctt cggcggcccc ccttgggggc gggcaggaat cccagggcgt tgcgggggtc  960ccggctgcgg gtgtgggggc cgccaccgcc ccctcccgcc tgcgtccgcg ccggcttccg 1020catctgctcg gcggcctcct ctgcgtctgg ctgtctcccc ccacttgcgt ctctctcccc 1080ccctttgttc tcgcctccga gcgctccccg cagcctcccc tcccccctgg tatttaaatc 1140gcctgcaggc ccggagccct ccccccgcgg gcctccgggg acacgcagtg tccatcccag 1200tggaggggcc catcggggga ggggcggagg gggagggtct cctttgtctg cgcggcggcg 1260gccgcctgcg ccggggaggg aggaggaggg ggagcccggc ccggcgcaac ccccagggcc 1320tctcctcggg ccgaaac                                                1337Sequence entry ends.

EXAMPLE 10

Example of Sequence Data Example of sequences submitted:

Sequence entry starts:SEQ_ID 1168 = Transcript NM_019103, corresponding to gene ZMAT5, as described in Table 4: <210> 1668 <211> 36025 <212> DNA<213> Homo sapiens <400> 1668tgtggtgaca gactttcttt ataaacattt ggaagttttc tcccccatct tcttaagaag    60caggggggca ggtggaggag agtgagggga gagctgcccg gtgcagaccc aggacgaggg   120ctgcacttgg tgtggccgtg tcctgagcct cagtgaggct gggcagatgg tctcggagcc   180... ... ... ... ... ... ...(sequence truncated for brevity)caagaaatgg tgcgtcccgc cgcagggcgt acgcacagag aaggaagtgt tcaagtcttc 35940cagtgcggag aaaagagact aggactcgcc cctcgacgtc tcgcggaagg tacctggctc 36000cccggtggct gcagctccgg gctcc                                       36025Sequence entry ends.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US11618925B2).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method of hybridizing a nucleic acid probe orsynthesizing a nucleic acid product comprising: (a) hybridizing anucleic acid probe to a polynucleic acid by nucleic acid hybridizationor microarray analysis, or synthesizing a nucleic acid product from apolynucleic acid by PCR or sequencing wherein the polynucleic acid isfrom a sample from a human subject that has Autism Spectrum Disorder(ASD); and (b) detecting a genetic variation in (i) the polynucleic acidby nucleic acid hybridization or microarray analysis or (ii) the nucleicacid product by PCR or sequencing, wherein the genetic variation is acopy number variant (CNV), wherein the CNV is a gain of SEQ ID NO: 701or the complement thereof.
 2. The method of claim 1, wherein the nucleicacid product synthesized from the polynucleic acid comprises cDNA. 3.The method of claim 1, wherein the polynucleic acid comprises a nucleicacid from blood, saliva, urine, serum, tears, skin, tissue, or hair fromthe subject.
 4. The method of claim 1, wherein the method comprisesisolating polynucleotides; and performing a microarray analysis of thepolynucleotides.
 5. The method of claim 1, wherein the microarrayanalysis is selected from the group consisting of a Comparative GenomicHybridization (CGH) array analysis and an SNP array analysis.
 6. Themethod of claim 5, wherein the microarray analysis comprises ComparativeGenomic Hybridization (CGH) array analysis.
 7. The method of claim 1,wherein the sequencing comprises high throughput sequencing.
 8. Themethod of claim 1, wherein the whole genome of the subject is analyzed.9. The method of claim 1, wherein the whole exome of the subject isanalyzed.
 10. The method of claim 1, wherein the detecting comprisesdetecting a first genetic variation that is the CNV that is SEQ ID NO:701 or the complement thereof, wherein the first genetic variation and asecond genetic variation are in a panel comprising two or more geneticvariations.
 11. The method of claim 10, wherein the panel comprises 50or more genetic variations.
 12. The method of claim 10, wherein thepanel comprises 100 or more genetic variations.
 13. The method of claim1, wherein the analysis comprises an in-silico analysis.
 14. A method oftreatment comprising administering an agent having a therapeutic benefitfor treatment of ASD, to a subject in need thereof, wherein the subjecthas been identified as having the genetic variation according to themethod of claim
 1. 15. A method of hybridizing a nucleic acid probe orsynthesizing a nucleic acid product comprising: (a) hybridizing anucleic acid probe to a polynucleic acid by nucleic acid hybridizationor microarray analysis, or synthesizing a nucleic acid product from apolynucleic acid by PCR or sequencing wherein the polynucleic acid isfrom a sample from a human subject that has Autism Spectrum Disorder(ASD); and (b) detecting a genetic variation in (i) the polynucleic acidby nucleic acid hybridization or microarray analysis or (ii) the nucleicacid product by PCR or sequencing, wherein the genetic variation is acopy number variant (CNV), wherein the CNV is a gain of SEQ ID NO: 702or the complement thereof.
 16. The method of claim 15, wherein themethod comprises isolating polynucleotides; and performing a microarrayanalysis of the polynucleotides.
 17. The method of claim 15, wherein themicroarray analysis is selected from the group consisting of aComparative Genomic Hybridization (CGH) array analysis and an SNP arrayanalysis.
 18. The method of claim 17, wherein the microarray analysiscomprises Comparative Genomic Hybridization (CGH) array analysis. 19.The method of claim 15, wherein the sequencing comprises high throughputsequencing.
 20. The method of claim 15, wherein the whole genome of thesubject is analyzed.
 21. The method of claim 15, wherein the whole exomeof the subject is analyzed.
 22. The method of claim 15, wherein thedetecting comprises detecting a first genetic variation that is the CNVthat is SEQ ID NO: 701 or the complement thereof, wherein the firstgenetic variation and a second genetic variation are in a panelcomprising two or more genetic variations.
 23. The method of claim 22,wherein the panel comprises 50 or more genetic variations.
 24. Themethod of claim 23, wherein the panel comprises 100 or more geneticvariations.
 25. The method of claim 15, wherein the analysis comprisesan in-silico analysis.
 26. A method of treatment comprisingadministering an agent having a therapeutic benefit for treatment ofASD, to a subject in need thereof, wherein the subject has beenidentified as having the genetic variation according to the method ofclaim 15.